Novel fxr (nr1h4) binding and activity modulating compounds

ABSTRACT

The present invention relates to compounds which bind to the NR1H4 receptor (FXR) and act as agonists of FXR. The invention further relates to the use of the compounds for the preparation of a medicament for the treatment of diseases and/or conditions through binding of said nuclear receptor by said compounds and to a process for the synthesis of said compounds.

The present invention relates to compounds which bind to the NR1H4receptor (FXR) and act as agonists or modulators of FXR. The inventionfurther relates to the use of the compounds for the treatment and/orprophylaxis of diseases and/or conditions through binding of saidnuclear receptor by said compounds.

Multicellular organisms are dependent on advanced mechanisms ofinformation transfer between cells and body compartments. Theinformation that is transmitted can be highly complex and can result inthe alteration of genetic programs involved in cellular differentiation,proliferation, or reproduction. The signals, or hormones, are often lowmolecular weight molecules, such as peptides, fatty acid, or cholesterolderivatives.

Many of these signals produce their effects by ultimately changing thetranscription of specific genes. One well-studied group of proteins thatmediate a cell's response to a variety of signals is the family oftranscription factors known as nuclear receptors, hereinafter referredto often as “NR”. Members of this group include receptors for steroidhormones, vitamin D, ecdysone, cis and trans retinoic acid, thyroidhormone, bile acids, cholesterol-derivatives, fatty acids (and otherperoxisomal proliferators), as well as so-called orphan receptors,proteins that are structurally similar to other members of this group,but for which no ligands are known. Orphan receptors may be indicativeof unknown signalling pathways in the cell or may be nuclear receptorsthat function without ligand activation. The activation of transcriptionby some of these orphan receptors may occur in the absence of anexogenous ligand and/or through signal transduction pathways originatingfrom the cell surface (D. J. Mangelsdorf et al., Cell 1995, 83, 835; R.M. Evans, Mol. Endocrinol. 2005, 19, 1429).

In general, three functional domains have been defined in NRs. An aminoterminal domain is believed to have some regulatory function. It isfollowed by a DNA-binding domain hereinafter referred to as “DBD” whichusually comprises two zinc finger elements and recognizes a specificHormone Responsive Element hereinafter referred to as “HRE” within thepromoters of responsive genes. Specific amino acid residues in the “DBD”have been shown to confer DNA sequence binding specificity (M. Schenaand K. R. Yamamoto, Science 1988, 241, 965). A ligand-binding-domainhereinafter referred to as “LBD” is at the carboxy-terminal region ofknown NRs.

In the absence of hormone, the LBD appears to interfere with theinteraction of the DBD with its HRE. Hormone binding seems to result ina conformational change in the NR and thus opens this interference (A.M. Brzozowski et al., Nature 1997, 389, 753). A NR without the LBDconstitutively activates transcription but at a low level.

Coactivators or transcriptional activators are proposed to bridgebetween sequence specific transcription factors, the basal transcriptionmachinery and in addition to influence the chromatine structure of atarget cell. Several proteins like SRC-1, ACTR, and Grip1 interact withNRs in a ligand enhanced manner (D. M. Heery et al., Nature 1997, 387,733; T. Heinzel et al., Nature 1997, 387, 43; K. W. Nettles and G. L.Greene, Annu. Rev. Physiol. 2005, 67, 309).

Nuclear receptor modulators like steroid hormones affect the growth andfunction of specific cells by binding to intracellular receptors andforming nuclear receptor-ligand complexes. Nuclear receptor-hormonecomplexes then interact with a HRE in the control region of specificgenes and alter specific gene expression (A. Aranda and A. Pascual,Physiol. Rev. 2001, 81, 1269).

The Farnesoid X Receptor alpha (hereinafter also often referred to asNR1H4 when referring to the human receptor) is a prototypical type 2nuclear receptor which activates genes upon binding to promoter regionof target genes in a heterodimeric fashion with Retinoid X Receptor (B.M. Forman et al., Cell 1995, 81, 687). The relevant physiologicalligands of NR1H4 are bile acids (D. J. Parks et al., Science 1999, 284,1365; M. Makishima et al., Science 1999, 284, 1362). The most potent oneis chenodeoxycholic acid (CDCA), which regulates the expression ofseveral genes that participate in bile acid homeostasis. Farnesol andderivatives, together called farnesoids, are originally described toactivate the rat orthologue at high concentration but they do notactivate the human or mouse receptor. FXR is expressed in the liver,throughout the entire gastrointestinal tract including the esophagus,stomach, duodenum, small intestine, colon, ovary, adrenal gland andkidney. Beyond controlling intracellular gene expression, FXR seems tobe also involved in paracrine and endocrine signalling by upregulatingthe expression of the cytokine Fibroblast Growth Factor 15 (rodents) or19 (monkeys, humans, J. A. Holt et al., Genes Dev. 2003, 17, 1581; T.Inagaki et al., Cell Metab. 2005, 2, 217).

Small molecule compounds which act as FXR modulators have been disclosedin the following publications: WO 2000/037077, WO 2003/015771, WO2004/048349, WO 2007/076260, WO 2007/092751, WO 2007/140174, WO2007/140183, WO 2008/051942, WO 2008/157270, WO 2009/005998, WO2009/012125, WO 2008/025539 and WO 2008/025540. Further small moleculeFXR modulators have been recently reviewed (M. L. Crawley, Expert OpinTher. Pat. 2010, 20,1047; D. Merk et al., Future Med. Chem. 2012, 4,1015). In WO 2011/020615 we disclosed chiral cyclopropylidene compoundsof the following general formula

wherein the variables are defined similar as in this application.

The problem underlying the present invention is to generate FXR-agonistswith improved physicochemical properties in general, and reducedhydrophobicity, improved aqueous solubility and better membranepermeability, in particular, compared to compounds claimed in WO2011/020615.

Said problem has been solved by a compound according to the followingFormula (1), an enantiomer, diastereomer, tautomer, solvate, prodrug orpharmaceutical acceptable salt thereof

wherein

R is selected from the group consisting of COOR₆, CONR₇R₈, tetrazolyl,SO₂NR₇R₈, C₁₋₆ alkyl, SO₂—C₁₋₆ alkyl and H, with R₆ independentlyselected from the group consisting of H or C₁₋₆ alkyl, and R₇ and R₈independently from each other selected from the group consisting of H,C₁₋₆ alkyl, halo-C₁₋₆ alkyl, C₁₋₆ alkylene-R₉, SO₂—C₁₋₆ alkyl, whereinR₉ is selected from the group consisting of COOH, OH and SO₃H;

A is selected from the group consisting of phenyl, pyridyl, pyrimidyl,pyrazolyl, indolyl, thienyl, benzothienyl, indazolyl, benzisoxazolyl,benzofuranyl, benzotriazolyl, furanyl, benzothiazolyl, thiazolyl,oxadiazolyl, each optionally substituted with one or two groupsindependently selected from the group consisting of OH, O—C₁₋₆ alkyl,O-halo-C₁₋₆ alkyl, C₁₋₆ alkyl, halo-C₁₋₆ alkyl, C₃₋₆ cycloalkyl andhalogen;

Q is selected from the group consisting of phenyl, pyridyl, thiazolyl,thiophenyl, pyrimidyl, each optionally substituted with one or twogroups independently selected from the group consisting of C₁₋₆ alkyl,halo-C₁₋₆ alkyl, halogen and CF₃;

Y is selected from N or CH;

Z is selected from

wherein

X═CH, N, NO;

R₁ is selected from the group consisting of hydrogen, C₁₋₃ alkyl, C₃₋₆cylcoalkyl, C₄₋₅ alkylcycloalkyl, wherein C₁₋₃ alkyl is optionallysubstituted with 1 to 3 substituents independently selected fromhalogen, hydroxy or C₁₋₆ alkoxy;

R₂ and R₃ are independently selected from the group consisting ofhydrogen, C₁₋₃ alkyl, C₁₋₃ haloalkyl, C₁₋₃ alkoxy, C₁₋₃ haloalkoxy andhalogen.

In another embodiment in combination with any of the above or belowembodiments, R-A in the compound according to Formula (1) is selectedfrom

In another embodiment in combination with any of the above or belowembodiments, Q in the compound according to Formula (1) is

In another embodiment in combination with any of the above or belowembodiments, Z in the compound according to Formula (1) is

In another embodiment in combination with any of the above or belowembodiments, the compound according to Formula (1) is selected from

In another embodiment in combination with any of the above or belowembodiments, the compound according to Formula (1) is

wherein R is selected from the group consisting of CO₂H, CONHSO₂Me, andtetrazolyl.

In another embodiment, the present invention is directed to a compoundaccording to Formula (1) for use as a medicament.

In another embodiment, the present invention is directed to a compoundaccording to Formula (1) for use in the prophylaxis and/or treatment ofdiseases mediated by FXR.

In another embodiment, the present invention is directed to the use of acompound according to Formula (1) for the preparation of a medicamentfor the prophylaxis and/or treatment of diseases mediated by FXR.

In another embodiment in combination with any of the above or belowembodiments, the disease is selected from chronic intrahepatic or someforms of extrahepatic cholestatic conditions; liver fibrosis;obstructive or chronic inflammatory disorders of the liver; livercirrhosis;

liver steatosis and associated syndromes, cholestatic or fibroticeffects that are associated with alcohol-induced cirrhosis or withviral-borne forms of hepatitis; liver failure or liver ischemia aftermajor liver resection; chemotherapy associated steatohepatitis (CASH);acute liver failure; and/or Inflammatory Bowel Diseases.

In another embodiment in combination with any of the above or belowembodiments, the disease is selected from lipid and lipoproteindisorders; Type II Diabetes and clinical complications of Type I andType II Diabetes, including diabetic nephropathy, diabetic neuropathy,diabetic retinopathy and other observed effects of clinically manifestlong term Diabetes; conditions and diseases which result from chronicfatty and fibrotic degeneration of organs due to enforced lipid andspecifically triglyceride accumulation and subsequent activation ofprofibrotic pathways, such as Non-Alcoholic Fatty Liver Disease (NAFLD),or Non-Alcoholic Steatohepatitis (NASH); obesity or metabolic syndrome(combined conditions of dyslipidemia, diabetes or abnormally highbody-mass index); and/or cute myocardial infarction, acute stroke orthrombosis which occurs as an endpoint of chronic obstructiveatherosclerosis.

In another embodiment in combination with any of the above or belowembodiments, the disease is selected from non-malignanthyperproliferative disorders and malignant hyperproliferative disorders,specifically of hepatocellular carcinoma, colon adenoma and polyposis,colon adenocarcinoma, breast cancer, pancreas adenocarcinoma, Barrett'sesophagus or other forms of neoplastic diseases of the gastrointestinaltract and the liver.

The improved physico-chemical properties have been achieved by theintroduction of a polar hydroxyl group on a 1,3-cyclobutylidene or1,3-azetidinylidene group replacing the former 1,2-cyclopropylidenering.

Surprisingly, the resulting compounds maintained their activity on theFXR receptor but demonstrated improved physico-chemical properties, suchas higher aqueous solubility and/or membrane permeability.

The compounds of the present invention share a common chemical structureaccording to Formula (1) in claim 1.

In a preferred embodiment in combination with any of the above or belowembodiments, the present invention is directed to an enantiomer,diastereomer or pharmaceutically acceptable salt of a compound accordingto Formula (1).

In a preferred embodiment in combination with any of the above or belowembodiments, R in Formula (1) is selected from the group consisting ofCOOR₆, CONR₇R₈, SO₂NR₇R₈, and SO₂—C₁₋₆ alkyl.

In a preferred embodiment in combination with any of the above or belowembodiments, R₆ in Formula (1) is H.

In a preferred embodiment in combination with any of the above or belowembodiments, R₇ and R₈ in Formula (1) are independently from each otherselected from the group consisting of H and SO₂—C₁₋₆ alkyl.

In a preferred embodiment in combination with any of the above or belowembodiments, R₇ in Formula (1) is H.

In a preferred embodiment in combination with any of the above or belowembodiments, R₈ in Formula (1) is SO₂—C₁₋₆ alkyl.

In a preferred embodiment in combination with any of the above or belowembodiments, A is selected from the group consisting of phenyl, pyridyl,pyrimidyl, pyrazolyl, indazolyl, and oxadiazolyl.

In a preferred embodiment in combination with any of the above or belowembodiments, A is substituted with one or two groups independentlyselected from C₁₋₆ alkyl, more preferably C₁₋₃ alkyl. In anotherpreferred embodiment in combination with any of the above or belowembodiments, A is unsubstituted.

In a preferred embodiment in combination with any of the above or belowembodiments, Q is phenyl.

In a preferred embodiment in combination with any of the above or belowembodiments, Q is substituted with one or two groups independentlyselected from halogen, more preferably one group selected from halogen,in particular Cl.

In a preferred embodiment in combination with any of the above or belowembodiments, Z is

In a preferred embodiment in combination with any of the above or belowembodiments, X═CH.

In a preferred embodiment in combination with any of the above or belowembodiments, R₁ is C3-6 cylcoalkyl, in particular cyclopropyl.

In a preferred embodiment in combination with any of the above or belowembodiments, R₂ and R₃ are independently selected from halogen, inparticular Cl.

The compounds of the present invention can be in the form of a prodrugcompound. “Prodrug compound” means a derivative that is converted into acompound according to the present invention by a reaction with anenzyme, gastric acid or the like under a physiological condition in theliving body, e.g. by oxidation, reduction, hydrolysis or the like, eachof which is carried out enzymatically. Examples of the prodrug arecompounds, wherein the amino group in a compound of the presentinvention is acylated, alkylated or phosphorylated to form, e.g.,eicosanoylamino, alanylamino, pivaloyloxymethylamino or wherein thehydroxyl group is acylated, alkylated, phosphorylated or converted intothe borate, e.g. acetyloxy, palmitoyloxy, pivaloyloxy, succinyloxy,fumaryloxy, alanyloxy or wherein the carboxyl group is esterified oramidated. These compounds can be produced from compounds of the presentinvention according to well-known methods. Other examples of the prodrugare compounds, wherein the carboxylate in a compound of the presentinvention is, for example, converted into an alkyl-, aryl-, choline-,amino, acyloxymethylester, linolenoylester.

Metabolites of compounds of the present invention are also within thescope of the present invention.

Where tautomerism, like e.g. keto-enol tautomerism, of compounds of thepresent invention or their prodrugs may occur, the individual forms,like e.g. the keto and enol form, are each within the scope of theinvention as well as their mixtures in any ratio. Same applies forstereoisomers, like e.g. enantiomers, cis/trans isomers, conformers andthe like.

If desired, isomers can be separated by methods well known in the art,e.g. by liquid chromatography. Same applies for enantiomers by usinge.g. chiral stationary phases. Additionally, enantiomers may be isolatedby converting them into diastereomers, i.e. coupling with anenantiomerically pure auxiliary compound, subsequent separation of theresulting diastereomers and cleavage of the auxiliary residue.Alternatively, any enantiomer of a compound of the present invention maybe obtained from stereoselective synthesis using optically pure startingmaterials. Another way to obtain pure enantiomers from racemic mixtureswould use enantioselective crystallization with chiral counterions.

The compounds of the present invention can be in the form of apharmaceutically acceptable salt or a solvate. The term“pharmaceutically acceptable salts” refers to salts prepared frompharmaceutically acceptable non-toxic bases or acids, includinginorganic bases or acids and organic bases or acids. In case thecompounds of the present invention contain one or more acidic or basicgroups, the invention also comprises their correspondingpharmaceutically or toxicologically acceptable salts, in particulartheir pharmaceutically utilizable salts. Thus, the compounds of thepresent invention which contain acidic groups can be present on thesegroups and can be used according to the invention, for example, asalkali metal salts, alkaline earth metal salts or ammonium salts. Moreprecise examples of such salts include sodium salts, potassium salts,calcium salts, magnesium salts or salts with ammonia or organic aminessuch as, for example, ethylamine, ethanolamine, triethanolamine or aminoacids. The compounds of the present invention which contain one or morebasic groups, i.e. groups which can be protonated, can be present andcan be used according to the invention in the form of their additionsalts with inorganic or organic acids. Examples of suitable acidsinclude hydrogen chloride, hydrogen bromide, phosphoric acid, sulfuricacid, nitric acid, methanesulfonic acid, p-toluenesulfonic acid,naphthalenedisulfonic acids, oxalic acid, acetic acid, tartaric acid,lactic acid, salicylic acid, benzoic acid, formic acid, propionic acid,pivalic acid, diethylacetic acid, malonic acid, succinic acid, pimelicacid, fumaric acid, maleic acid, malic acid, sulfaminic acid,phenylpropionic acid, gluconic acid, ascorbic acid, isonicotinic acid,citric acid, adipic acid, and other acids known to the person skilled inthe art. If the compounds of the present invention simultaneouslycontain acidic and basic groups in the molecule, the invention alsoincludes, in addition to the salt forms mentioned, inner salts orbetaines (zwitterions). The respective salts can be obtained bycustomary methods which are known to the person skilled in the art like,for example, by contacting these with an organic or inorganic acid orbase in a solvent or dispersant, or by anion exchange or cation exchangewith other salts. The present invention also includes all salts of thecompounds of the present invention which, owing to low physiologicalcompatibility, are not directly suitable for use in pharmaceuticals butwhich can be used, for example, as intermediates for chemical reactionsor for the preparation of pharmaceutically acceptable salts.

Further the compounds of the present invention may be present in theform of solvates, such as those which include as solvate water, orpharmaceutically acceptable solvates, such as alcohols, in particularethanol.

Furthermore, the present invention provides pharmaceutical compositionscomprising at least one compound of the present invention, or a prodrugcompound thereof, or a pharmaceutically acceptable salt or solvatethereof as active ingredient together with a pharmaceutically acceptablecarrier.

“Pharmaceutical composition” means one or more active ingredients, andone or more inert ingredients that make up the carrier, as well as anyproduct which results, directly or indirectly, from combination,complexation or aggregation of any two or more of the ingredients, orfrom dissociation of one or more of the ingredients, or from other typesof reactions or interactions of one or more of the ingredients.Accordingly, the pharmaceutical compositions of the present inventionencompass any composition made by admixing at least one compound of thepresent invention and a pharmaceutically acceptable carrier.

The pharmaceutical composition of the present invention may additionallycomprise one or more other compounds as active ingredients like aprodrug compound or other nuclear receptor modulators.

The compositions are suitable for oral, rectal, topical, parenteral(including subcutaneous, intramuscular, and intravenous), ocular(ophthalmic), pulmonary (nasal or buccal inhalation) or nasaladministration, although the most suitable route in any given case willdepend on the nature and severity of the conditions being treated and onthe nature of the active ingredient. They may be conveniently presentedin unit dosage form and prepared by any of the methods well-known in theart of pharmacy.

The compounds of the present invention can be prepared by a combinationof methods described in Schemes I to III. As depicted in Scheme I a4-membered cyclic ketone, substituted with substituent A in the3-position can react with a metalated aromatic or heteroaromatic ringM-Q-O—CH₂Z (M=metal, e.g. Li) in aprotic solvents and preferably at lowtemperatures to afford a hydroxyl substituted 4-membered ring bearingthe substituents A and Q. In the case where Y is CH two isomers can form(A and Q transannular cis or trans to each other). Under optimizedconditions the formation of mainly one of the two isomers can beachieved. The two isomers can be separated by appropriate methods knownin the art like e.g. silica gel chromatography or preparative RP-HPLC.

In Scheme II the methods are summarized which are used to prepare the4-membered cyclic ketones needed for the synthesis of the compounds ofthis invention. In option a) a vinyl bearing intermediate, e.g. preparedby vinylation of a corresponding halogen-containing starting materialR-A-X (X=halogen) can react with in situ formed α,α-dichloro ketene toform a 2,2-dichlorocyclobutanone. After dehalogenation, e.g. with Zn inacetic acid under reflux, the desired 3-substituted cyclobutanones areobtained. Alternatively, the vinyl-intermediates can react with in situgenerated unsubtituted ketene to afford in one step the desiredcyclobutanone intermediates. In option b)3-methylenecyclobutanecarbonitrile is used as starting material.Substituted heterocycles can be built up from the cyano group in severalsteps by methods known to those skilled in the art. The desiredcyclobutanones can be obtained by oxidative cleavage of the exocyclicdouble bond using conditions and reagents known to those skilled in theart, e.g. by the use of OsO₄, ozone or RhCl₃/NaIO₄ as oxidants. Optionc) shows the methods used to prepare the substituted azetidinones. Cu-or Pd-catalysed C—N cross coupling between 3-hydroxy-azetidine andhalo-aromatic or halo-heteroaromatic rings afford the correspondingN-substituted 3-hydroxy-azetidines which can be transformed into thedesired azetidinones by oxidation.

Scheme III illustrates some possibilities to perform modifications ofthe substituents at the A group after the formation of the 4-memberedhydroxy-bearing rings. For example, a leaving group X (e.g. bromide) canbe substituted by a cyano group, a carboxylic ester, methylsulfonyl orthioether by transition metal catalysed cross coupling reactions. Theobtained derivatives can be further transformed into other derivativesby methods known to those skilled in the art. For example, the cyano andthe ester group can be hydrolysed under basic conditions to the afford acarboxylic acid which in turn can be transformed into acyl-sulfonamides.A benzyl thioether can be chlorinated to afford the chlorosulfonylintermediate which reacts with ammonia to the correspondingsulfonamides.

As a result, the present invention relates to compounds according to thegeneral Formula (1) which bind to FXR and act as agonists or modulatorsof FXR.

The invention further relates to the use of said compounds for thetreatment and/or prophylaxis of diseases and/or conditions throughbinding of said nuclear receptor by said compounds. Further the presentinvention relates to the use of said compounds for the preparation of amedicament for the treatment and/or prophylaxis of diseases and/orconditions through binding of said nuclear receptor by said compounds.Specifically, the present invention relates to the use of compoundsaccording to Formula (1) in the preparation of a medicament for theprophylaxis and/or treatment of chronic intrahepatic or some forms ofextrahepatic cholestatic conditions, of liver fibrosis, of acuteintraheptic cholestatic conditions, of obstructive or chronicinflammatory disorders that arise out of improper bile composition, ofgastrointestinal conditions with a reduced uptake of dietary fat andfat-soluble dietary vitamins, of inflammatory bowel diseases, of lipidand lipoprotein disorders, of Type II Diabetes and clinicalcomplications of Type I and Type II Diabetes, of conditions and diseaseswhich result from chronic fatty and fibrotic degeneration of organs dueto enforced lipid and specifically triglyceride accumulation andsubsequent activation of profibrotic pathways, of obesity and metabolicsyndrome (combined conditions of dyslipidemia, diabetes and abnormallyhigh body-mass index), of acute myocardial infarction, of acute stroke,of thrombosis which occurs as an endpoint of chronic obstructiveatherosclerosis, of persistant infections by intracellular bacteria orparasitic protozoae, of non-malignant hyperproliferative disorders, ofmalignant hyperproliferative disorders, of colon adenocarcinoma andhepatocellular carcinoma in particular, of liver steatosis andassociated syndromes, of liver failure or liver malfunction as anoutcome of chronic liver diseases or of surgical liver resection, ofHepatitis B infection, of Hepatitis C infection and/or of cholestaticand fibrotic effects that are associated with alcohol-induced cirrhosisor with viral-borne forms of hepatitis.

Medicaments as referred to herein may be prepared by conventionalprocesses, including the combination of a compound according to thepresent invention and a pharmaceutically acceptable carrier.

FXR is proposed to be a nuclear bile acid sensor. As a result, itmodulates both, the synthetic output of bile acids in the liver andtheir recycling in the intestine (by regulating bile acid bindingproteins). But beyond bile acid physiology, FXR seems to be involved inthe regulation of many diverse physiological processes which arerelevant in the etiology and for the treatment of diseases as diverse ascholesterol gallstones, metabolic disorders such as Type II Diabetes,dyslipidemias or obesity, chronic inflammatory diseases such asInflammatory Bowel Diseases or chronic intrahepatic forms of cholestasisand many others diseases (T. Claudel et al., Arterioscler. Thromb. Vasc.Biol. 2005, 25, 2020; Y. D. Wang et al., Cell Res. 2008, 18, 1087.

FXR regulates a complex pattern of response genes in the liver and inthe gastrointestinal tract. The gene products have impact on diversephysiological processes. In the course of functional analysis of FXR,the first regulatory network that was analyzed was the regulation ofbile acid synthesis. While the LXRs induce the key enzyme of theconversion of cholesterol into bile acids, Cyp7A1, via the induction ofthe regulatory nuclear receptor LRH-1, FXR represses the induction ofCyp7A1 via the upregulation of mRNA encoding SHP, a further nuclearreceptor that is dominant repressive over LRH-1. Since FXR binds the endproducts of this pathway, primary bile acids such as cholic acid (CA) orCDCA, this can be regarded as an example of feedback inhibition on thegene expression level (B. Goodwin et al., Mol. Cell 2000, 6, 517; T. T.Lu et al., Mol. Cell 2000, 6, 507). Parallel to the repression of bileacid synthesis via SHP, FXR induces a range of so-called ABC (forATP-binding cassette) transporters that are responsible for the exportof toxic bile acids from the hepatocyte cytosol into the canaliculi, thesmall bile duct ramifications where the bile originates. Thishepatoprotective function of FXR became first apparent with the analysisof FXR knockout mice (C. J. Sinal et al., Cell 2000, 102, 731), whereunder- or overexpression of several ABC-transporters in the liver wasshown. Further detailed analysis revealed that the major bile saltexcretory pump BSEP or ABCB11 (M. Ananthanarayanan et al., J. Biol.Chem. 2001, 276, 28857; J. R. Plass et al., Hepatology 2002, 35, 589) aswell as the key enzyme which mediates lipid transfer from lipoproteinsto phospholipids, PLTP (N. L. Urizar et al., J. Biol. Chem. 2000, 275,39313), and the two key canalicular membrane transporters forphospholipids, MRP-2 (ABCC4) (H. R. Kast et al., J. Biol. Chem. 2002,277, 2908) and MDR-3 (ABCB4); L. Huang et al., J. Biol. Chem. 2003, 278,51085) are direct targets for ligand-directed transcriptional activationby FXR (summarized in: M. Miyata, J. Pharmacol. Exp. Ther. 2005, 312,759; G. Rizzo et al., Curr. Drug Targets Immune Endocr. Metabol. Disord.2005, 5, 289).

The fact that FXR seems to be the major metabolite sensor and regulatorfor the synthesis, export and re-circulation of bile acids suggested theuse of FXR ligands to induce bile flow and change bile acid compositiontowards more hydrophilic composition. With the development of the firstsynthetic FXR ligand GW4064 (P. R. Maloney et al., J. Med. Chem. 2000,43, 2971; T. M. Willson et al., Med. Res. Rev. 2001, 21, 513) as a toolcompound and of the semi-synthetic artificial bile acid ligand6-alpha-ethyl-CDCA, the effects of superstimulation of FXR by potentagonists could be analyzed. It was shown that both ligands induce bileflow in bile duct ligated animals. Moreover, in addition to cholereticeffects, also hepatoprotective effects could be demonstrated (R.Pellicciari et al., J. Med. Chem. 2002, 45, 3569; Y. Liu et al., J.Clin. Invest. 2003, 112, 1678). This hepatoprotective effect was furthernarrowed down to an anti-fibrotic effect that results from therepression of Tissue Inhibitors of Matrix-Metalloproteinases, TIMP-1 and2, the induction of collagen-deposit resolving Matrix-Metalloproteinase2 in hepatic stellate cells and the subsequent reduction ofalpha-collagen mRNA and Transforming growth factor beta (TGF-beta) mRNAwhich are both pro-fibrotic factors by FXR agonists (S. Fiorucci et al.,Gastroenterology 2004, 127, 1497; S. Fiorucci et al., J. Pharmacol. Exp.Ther. 2005, 314, 584). Furthermore, anti-cholestatic activity wasdemonstrated in bile-duct ligated animal models as well as in animalmodels of estrogen-induced cholestasis (S. Fiorucci et al., J.Pharmacol. Exp. Ther. 2005, 313, 604).

Genetic studies demonstrate that in hereditary forms of cholestasis(Progressive Familiar Intrahepatic Cholestasis=PFIC, Type I-IV) eithernuclear localization of FXR itself is reduced as a consequence of amutation in the FIC1 gene (in PFIC Type I, also called Byler's Disease)(F. Chen et al., Gastroenterology 2004, 126, 756; L. Alvarez et al.,Hum. Mol. Genet. 2004, 13, 2451) or levels of the FXR target geneencoding MDR-3 phospholipid export pump are reduced (in PFIC Type III).Taken together there is a growing body of evidence that FXR bindingcompounds will demonstrate substantial clinical utility in thetherapeutic regimen of chronic cholestatic conditions such as PrimaryBiliary Cirrhosis (PBC) or Primary Sclerosing Cholangitis (PSC)(reviewed in: G. Rizzo et al., Curr. Drug Targets Immune Endocr.Metabol. Disord. 2005, 5, 289; G. Zollner et al., Mol. Pharm. 2006, 3,231; S. Y. Cai et al., Expert Opin. Ther. Targets 2006, 10, 409).

The deep impact that FXR activation has on bile acid metabolism andexcretion is not only relevant for cholestatic syndromes but even moredirectly for a therapy against gallstone formation. Cholesterolgallstones form due to low solubility of cholesterol that is activelypumped out of the liver cell into the lumen of the canaliculi. It is therelative percentage of content of the three major components, bileacids, phospholipids and free cholesterol that determines the formationof mixed micelles and hence apparent solubility of free cholesterol inthe bile. FXR polymorphisms map as quantitative trait loci as one factorcontributing to gallstone disease (H. Wittenburg, Gastroenterology 2003,125, 868). Using the synthetic FXR tool compound GW4064 it could bedemonstrated that activation of FXR leads to an improvement of theCholesterol Saturation Index (CSI) and directly to an abolishment ofgallstone formation in C57L gallstone susceptible mice whereas drugtreatment in FXR knockout mice shows no effect on gallstone formation(A. Moschetta et al., Nature Medicine 2004, 10, 1352).

These results qualify FXR as a good target for the development of smallmolecule agonists that can be used to prevent cholesterol gallstoneformation or to prevent re-formation of gallstones after surgicalremoval or shockwave lithotripsy (discussed in: S. A. Doggrell, Curr.Opin. Investig. Drugs 2006, 7, 344).

Thus, in one embodiment of the invention, the compound according toFormula (1) and pharmaceutical compositions comprising said compound isused for the prophylaxis and/or treatment of obstructive or chronicinflammatory disorders that arise out of improper bile composition suchas cholelithiasis also known as cholesterol gallstones.

Beyond its strong hepatoprotective and choleretic as well asanti-fibrotic effects that FXR shows upon small molecule stimulatedactivation in the liver, FXR seems to have a role in protecting theintestine from neoplastic transformation and from the development ofpolyps and their transition into adenocarcinoma in the gut (S. Modica etal., Cancer Res. 2008, 68, 9589 and R. R. Maran et al., J. Pharmacol.Exp. Ther. 2009, 328, 469). Similar to the situation in the intestineabsence of FXR leads to a high increase in the formation ofHepatocellular Cacrcinoma (HCC), the most prominent form of liver cancer(I. Kim et al., Carcinogenesis 2007, 28, 940 and F. Yang et al., CancerRes. 2007, 67, 863). Whereas a functional FXR prevents the formation ofcolon adenocarcinoma and hepatocellular carcinoma, FXR activationinduces liver regeneration after hepatectomy (W. Huang et al., Science2006, 312, 233).

The combined hepatoprotective, anti-neoplastic and liver regenerativeeffects associated with FXR activation can be therapeutically exploitedfor the use of FXR agonists in the treatment of sever liver diseases. Inone embodiment, the compounds according to the invention andpharmaceutical compositions comprising said compounds are used in thetreatment of liver diseases such as HCC, stimulation of liver regrowthand amelioration of side effects associated with major liver resection,liver cirrhosis independent of the etiology and prevention or treatmentof liver ischemia in the course of liver transplantation or major liversurgery.

Since the discovery of the first synthetic FXR agonist and itsadministration to rodents it became evident that FXR is a key regulatorof serum triglycerides (P. Maloney et al., J. Med. Chem. 2000, 43, 2971;T. Willson et al., Med. Res. Rev. 2001, 21, 513). Over the past sixyears accumulating evidence has been published that activation of FXR bysynthetic agonists leads to significant reduction of serumtriglycerides, mainly in the form of reduced VLDL, but also to reducedtotal serum cholesterol (H. R. Kast et al., Mol. Endocrinol. 2001, 15,1720; N. L. Urizar et al., Science 2002, 296, 1703; G. Lambert et al.,J. Biol. Chem. 2003, 278, 2563; M. Watanabe et al., J. Clin. Invest.2004, 113, 1408; A. Figge et al., J. Biol. Chem. 2004, 279, 2790; S.Bilz et al., Am. J. Physiol. Endocrinol. Metab. 2006, 290, E716).

But the lowering of serum triglycerides is not a stand alone effect.Treatment of db/db or ob/ob mice with synthetic FXR agonist GW4064resulted in marked and combined reduction of serum triglycerides, totalcholesterol, free fatty acids, ketone bodies such as 3-OH Butyrate.Moreover, FXR activation engages with the intracellular insulinsignaling pathway in hepatocytes, resulting in reduced output of glucosefrom liver gluconeogenesis but concomitant increase in liver glycogen.Insulin sensitivity as well as glucose tolerance were positivelyimpacted by FXR treatment (K. R. Stayrook et al., Endocrinology 2005,146, 984; Y. Zhang et al., PNAS 2006, 103, 1006; B. Cariou et al., J.Biol. Chem. 2006, 281, 11039; K. Ma et al., J. Clin. Invest. 2006, 116,1102; D. Duran-Sandoval et al., Biochimie 2005, 87, 93). An effect onreduction of body weight was also recently observed in mice overfed witha high lipid diet (C. Lihong et al., American Diabetes Association (ADA)66^(th) annual scientific sessions, June 2006, Abstract Number 856-P).This weight loss effect might results from FXR's induction of FGF-19, afibroblast growth factor that is known to lead to weight loss andathletic phenotype (J. Holt et al., Genes Dev. 2003, 17, 1581; E.Tomlinson et al., Endocrinology 2002, 143, 1741). In recent patentapplications, the effect of FXR agonist on reduction of body weight wasdemonstrated (WO 2004/087076; WO 2003/080803).

Taken together, these pharmacological effects of FXR agonists can beexploited in different therapeutic ways: FXR binding compounds arethought to be good candidates for the treatment of Type II Diabetesbecause of their insulin sensitization, glycogenogenic, and lipidlowering effects.

In one embodiment, the compounds according to the invention andpharmaceutical compositions comprising said compounds are used in theprophylaxis and/or treatment of Type II Diabetes which can be overcomeby FXR-mediated upregulation of systemic insulin sensitivity andintracellular insulin signalling in liver, increased peripheral glucoseuptake and metabolisation, increased glycogen storage in liver,decreased output of glucose into serum from liver-borne gluconeogenesis.

In a further embodiment, said compounds and pharmaceutical compositionsare used for the prophylaxis and/or treatment of chronic intrahepatic,such as PBC, PSC, progressive familiar cholestasis (PFIC),alcohol-induced cirrhosis and associated cholestasis, and some forms ofextrahepatic cholestatic conditions, or liver fibrosis.

The invention also relates to a compound of Formula (1) or to apharmaceutical composition comprising said compound for the prophylaxisand/or treatment of gastrointestinal conditions with a reduced uptake ofdietary fat and fat-soluble dietary vitamins which can be overcome byincreased intestinal levels of bile acids and phospholipids.

In a further embodiment, said compound or pharmaceutical composition isused for preventing and/or treating a disease selected from the groupconsisting of lipid and lipoprotein disorders such ashypercholesterolemia, hypertriglyceridemia, and atherosclerosis as aclinically manifest condition which can be ameliorated by FXR'sbeneficial effect on lowering total plasma cholesterol, lowering serumtriglycerides, increasing conversion of liver cholesterol into bileacids and increased clearance and metabolic conversion of VLDL and otherlipoproteins in the liver.

In one further embodiment, said compound and pharmaceutical compositionare used for the prophylaxis and/or treatment of diseases where thecombined lipid lowering, anti-cholestatic and anti-fibrotic effects ofFXR-targeted medicaments can be exploited for the treatment of liversteatosis and associated syndromes such as NASH, or for the treatment ofcholestatic and fibrotic effects that are associated withalcohol-induced cirrhosis, or with viral-borne forms of hepatitis.

In conjunction with the hypolipidemic effects it was also shown thatloss of functional FXR leads to increased atherosclerosis in ApoEknockout mice (E. A. Hanniman et al., J. Lipid Res. 2005, 46, 2595).Therefore, FXR agonists might have clinical utility asanti-atherosclerotic and cardioprotective drugs. The downregulation ofEndothelin-1 in Vascular Smooth Muscle Cells might also contribute tosuch beneficial therapeutic effects (F. He et al., Circ. Res. 2006, 98,192).

The invention also relates to a compound according to Formula (1) or apharmaceutical composition comprising said compound for preventive andposttraumatic treatment of cardiovascular disorders such as acutemyocardial infarction, acute stroke, or thrombosis which occur as anendpoint of chronic obstructive atherosclerosis.

Beyond controlling intestinal and colonic polyp formation, FXR seems tobe expressed in breast cancer tissue and cell lines but not in healthybreast tissue and seems to interact with the Estrogen Receptor in ERpositive breast cancer cells (K. E. Swales et al., Cancer Res. 2006, 66,10120 and F. Journe et al., Breast Cancer Res. Treat. 2009, 115, 523).

This would allow to regard FXR also as a potential target for thetreatment of proliferative diseases, especially metastasizing cancerforms that express a small molecule responsive form of FXR.

In a further embodiment, said compounds and pharmaceutical compositionsare used for the prophylaxis and/or treatment of malignanthyperproliferative disorders such as different forms of cancer,specifically certain forms of breast, liver or colon cancer whereinterference with an FXR ligand will have a beneficial impact.

Finally, FXR seems also to be involved in the control of antibacterialdefense in the intestine (T. lnagaki et al., PNAS. 2006, 103, 3920)although an exact mechanism is not provided. From these published data,however, one can conclude that treatment with FXR agonists might have abeneficial impact in the therapy of Inflammatory Bowel Disorders (IBD),in particular those forms where the upper (Heal) part of the intestineis affected (e.g. ileal Crohn's disease) because this seems to be thesite of action of FXR's control on bacterial growth. In IBD thedesensitization of the adaptive immune response is somehow impaired inthe intestinal immune system. Bacterial overgrowth might then be thecausative trigger towards establishment of a chronic inflammatoryresponse. Hence, dampening of bacterial growth by FXR-borne mechanismsmight be a key mechanism to prevent acute inflammatory episodes. Thus,the invention also relates to a compound according to Formula (1) or apharmaceutical composition comprising said compound for preventingand/or treating a disease related to Inflammatory Bowel Diseases such asCrohn's disease or Colitis ulcerosa. FXR-mediated restoration ofintestinal barrier function and reduction in non-commensal bacterialload is believed to be helpful in reducing the exposure of bacterialantigens to the intestinal immune system and can therefore reduceinflammatory responses.

The invention further relates to a compound or pharmaceuticalcomposition for the prophylaxis and/or treatment of obesity andassociated disorders such as metabolic syndrome (combined conditions ofdyslipidemias, diabetes and abnormally high body-mass index) which canbe overcome by FXR-mediated lowering of serum triglycerides, bloodglucose and increased insulin sensitivity and FXR-mediated weight loss.

In a further embodiment, the compounds or pharmaceutical composition ofthe present invention are useful in preventing and/or treating clinicalcomplications of Type I and Type II Diabetes. Examples of suchcomplications include Diabetic Nephropathy, Diabetic Retinopathy,Diabetic Neuropathies, or Peripheral Arterial Occlusive Disease (PAOD).Other clinical complications of Diabetes are also encompassed by thepresent invention.

Furthermore, conditions and diseases which result from chronic fatty andfibrotic degeneration of organs due to enforced lipid and specificallytriglyceride accumulation and subsequent activation of profibroticpathways may also be prevented and/or treated by applying the compoundsor pharmaceutical composition of the present invention. Such conditionsand diseases encompass NASH and chronic cholestatic conditions in theliver, Glomerulosclerosis and Diabetic Nephropathy in the kidney, MaculaDegeneration and Diabetic Retinopathy in the eye and Neurodegenerativediseases such as Alzheimer's Disease in the brain, or DiabeticNeuropathies in the peripheral nervous system.

In practical use, the compounds of the present invention can be combinedas the active ingredient in intimate admixture with a pharmaceuticalcarrier according to conventional pharmaceutical compounding techniques.The carrier may take a wide variety of forms depending on the form ofpreparation desired for administration, e.g., oral or parenteral(including intravenous). In preparing the compositions for oral dosageform, any of the usual pharmaceutical media may be employed, such as,for example, water, glycols, oils, alcohols, flavoring agents,preservatives, coloring agents and the like in the case of oral liquidpreparations, such as, for example, suspensions, elixirs and solutions;or carriers such as starches, sugars, microcrystalline cellulose,diluents, granulating agents, lubricants, binders, disintegrating agentsand the like in the case of oral solid preparations such as, forexample, powders, hard and soft capsules and tablets, with the solidoral preparations being preferred over the liquid preparations.

Because of their ease of administration, tablets and capsules representthe most advantageous oral dosage unit form in which case solidpharmaceutical carriers are obviously employed. If desired, tablets maybe coated by standard aqueous or non-aqueous techniques. Suchcompositions and preparations should contain at least 0.1 percent ofactive compound. The percentage of active compound in these compositionsmay, of course, be varied and may conveniently be between about 2percent to about 60 percent of the weight of the unit. The amount ofactive compound in such therapeutically useful compositions is such thatan effective dosage will be obtained. The active compounds can also beadministered intranasally as, for example, liquid drops or spray.

The tablets, pills, capsules, and the like may also contain a bindersuch as gum tragacanth, acacia, corn starch or gelatin; excipients suchas dicalcium phosphate; a disintegrating agent such as corn starch,potato starch, alginic acid; a lubricant such as magnesium stearate; anda sweetening agent such as sucrose, lactose or saccharin. When a dosageunit form is a capsule, it may contain, in addition to materials of theabove type, a liquid carrier such as a fatty oil.

Various other materials may be present as coatings or to modify thephysical form of the dosage unit. For instance, tablets may be coatedwith shellac, sugar or both. A syrup or elixir may contain, in additionto the active ingredient, sucrose as a sweetening agent, methyl andpropylparabens as preservatives, a dye and a flavoring such as cherry ororange flavor.

Since the compounds of the present invention mostly represent carboxylicacids or similar anionic isosters thereof, and since it is well knownthat salt forms of ionic drug compounds can substantially affect thebioavailability of drug compounds, the compounds of the presentinvention may also be used as salts with various countercations to yieldan orally available formulation. Such pharmaceutically acceptablecations may be amongst others mono- or bivalent ions such as ammonium,the alkaline metals sodium or potassium or the alkaline earth metalsmagnesium or calcium, certain pharmaceutically acceptable amines such astris(hydroxymethyl)aminomethane, ethylendiamine, diethylamine,piperazine or others, or certain cationic amino acids such as lysine orarginine.

The compounds of the present invention may also be administeredparenterally. Solutions or suspensions of these active compounds can beprepared in water suitably mixed with a surfactant such ashydroxy-propylcellulose. Dispersions can also be prepared in glycerol,liquid polyethylene glycols and mixtures thereof in oils. Under ordinaryconditions of storage and use, these preparations contain a preservativeto prevent the growth of microorganisms.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases, the form must be sterile and must be fluid tothe extent that easy syringability exists. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (e.g., glycerol, propylene glycol and liquidpolyethylene glycol), suitable mixtures thereof, and vegetable oils.

Any suitable route of administration may be employed for providing amammal, especially a human, with an effective dose of a compound of thepresent invention. For example, oral, rectal, topical, parenteral,ocular, pulmonary, nasal, and the like may be employed. Dosage formsinclude tablets, troches, dispersions, suspensions, solutions, capsules,creams, ointments, aerosols, and the like. Preferably compounds of thepresent invention are administered orally.

The effective dosage of active ingredient employed may vary depending onthe particular compound employed, the mode of administration, thecondition being treated and the severity of the condition being treated.Such dosage may be ascertained readily by a person skilled in the art.

When treating or preventing FXR mediated conditions for which compoundsof the present invention are indicated, generally satisfactory resultsare obtained when the compounds of the present invention areadministered at a daily dosage of from about 0.1 milligram to about 100milligram per kilogram of animal body weight, preferably given as asingle daily dose or in divided doses two to six times a day, or insustained release form. For most large mammals, the total daily dosageis from about 1.0 milligrams to about 1000 milligrams, preferably fromabout 1 milligram to about 50 milligrams. In the case of a 70 kg adulthuman, the total daily dose will generally be from about 7 milligrams toabout 350 milligrams. This dosage regimen may be adjusted to provide theoptimal therapeutic response.

The compounds of the present invention can be prepared according to theprocedures of the following Schemes and Examples, using appropriatematerials and are further exemplified by the following specificexamples. Moreover, by utilizing the procedures described herein, inconjunction with ordinary skills in the art, additional compounds of thepresent invention claimed herein can be readily prepared. The compoundsillustrated in the examples are not, however, to be construed as formingthe only genus that is considered as the invention. The examples furtherillustrate details for the preparation of the compounds of the presentinvention. Those skilled in the art will readily understand that knownvariations of the conditions and processes of the following preparativeprocedures can be used to prepare these compounds. The instant compoundsare generally isolated in the form of their pharmaceutically acceptablesalts, such as those described above.

The amine-free bases corresponding to the isolated salts can begenerated by neutralization with a suitable base, such as aqueous sodiumhydrogen carbonate, sodium carbonate, sodium hydroxide and potassiumhydroxide, and extraction of the liberated amine-free base into anorganic solvent, followed by evaporation. The amine-free base, isolatedin this manner, can be further converted into another pharmaceuticallyacceptable salt by dissolution in an organic solvent, followed byaddition of the appropriate acid and subsequent evaporation,precipitation or crystallization. The carboxylic free acidscorresponding to the isolated salts can be generated by neutralizationwith a suitable acid, such as aqueous hydrochloric acid, sodium hydrogensulfate, sodium dihydrogen phosphate, and extraction of the liberatedcarboxylic-free acid into an organic solvent, followed by evaporation.The carboxylic acid, isolated in this manner, can be further convertedinto another pharmaceutically acceptable salt by dissolution in anorganic solvent, followed by addition of the appropriate base andsubsequent evaporation, precipitation or crystallization.

An illustration of the preparation of compounds of the present inventionis shown below. Unless otherwise indicated in the schemes, the variableshave the same meaning as described above.

The examples presented below are intended to illustrate particularembodiments of the invention. Suitable starting materials, buildingblocks and reagents employed in the synthesis as described below arecommercially available from Sigma-Aldrich or Acros Organics, forexample, or can be routinely prepared by procedures described in theliterature, for example in “March's Advanced Organic Chemistry:Reactions, Mechanisms, and Structure”, 5^(th) Edition; John Wiley & Sonsor T. Eicher, S. Hauptmann “The Chemistry of Heterocycles; Structures,Reactions, Synthesis and Application”, 2^(nd) edition, Wiley-VCH 2003;Fieser et al. “Fiesers' Reagents for organic Synthesis” John Wiley &Sons 2000.

EXAMPLES Examples Methyl3-((1s,3s)-3-(2-chloro-4-((5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazol-4-yl)methoxy)phenyl)-3-hydroxycyclobutyl)benzoate(1)

Step 1:4-((4-Bromo-3-chlorophenoxy)methyl)-5-cyclopropyl-3-(2,6-dichlorophenyl)-isoxazole(1a)

To a solution of(5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazol-4-yl)methanol (13 g, 45.8mmol) in CH₂Cl₂ (DCM) (200 mL) was added dropwise SOCl₂ (40 mL, 336mmol). The resulting mixture was stirred at rt for 2 h and the solventswere removed under reduced pressure. The residue was dissolved inN,N-dimethylformamide (DMF) (200 ml) and 4-bromo-3-chlorophenol (9.7 g,47 mmol), K₂CO₃ (40 g, 290 mmol) and NaI (12 g, 80 mmol) were added tothis solution. The mixture was stirred at 60° C. overnight, then cooledto rt, diluted with water (1000 mL) and extracted with ethyl acetate(EA) (500 mL×3). The combined organic phases were washed with brine (500mL×3), dried over Na₂SO₄ and concentrated in vacuo. The residue waspurified by flash chromatography on silica gel (CC) to give the titlecompound 1a (19 g, 88%) as a white solid.

Step 1: Methyl 3-(2,2-dichloro-3-oxocyclobutyl)benzoate (1 b)

To a 3-necked round bottomed flask, under a nitrogen atmosphere, fittedwith a condenser, an overhead stirrer and pressure equalised droppingfunnel was dissolved methyl 3-vinylbenzoate (5 g, 31 mmol) in dry Et₂O(150 mL). To this flask was added zinc dust (6 g, 3 eq) and the reactionwas sonicated for 30 min. After this time a solution oftrichloroacetylchloride (8.7 mL, 2.5 eq) in dry Et₂O (50mL) was addeddropwise whilst continuing the sonication over the next 30 min. Duringthe process the reaction mixture was heated to 35° C. The sonication wascontinued for 2.5 h at reflux and the reaction appeared to be completeby ¹H NMR analysis. The reaction was allowed to cool to rt and quenchedwith water (˜50 mL). This was done in a dropwise manner interspersedseveral times by a few minutes since a delayed exothermic reactionoccurred. After 20 min stirring in water the reaction mixture wasfiltered through a pad of celite and rinsed through with Et₂O. Theorganic layer was washed with portions of water (2×250 mL), saturatedsodium bicarbonate (2×250 mL) and brine (1×250 mL), dried over sodiumsulfate, filtered and concentrated under reduced pressure to afford thecrude product 1b as a dark yellow thick oil (crude 8.7 g).

Step 2: Methyl 3-(3-oxocyclobutyl)benzoate (1c)

Crude compound 1b (8.7 g) was dissolved in glacial acetic acid (55 mL)in a round bottomed flask under a nitrogen atmosphere. To this flask wasadded zinc dust (4.6 g, 2.2 eq) and the reaction was stirred and heatedto 120° C. for 3 h. After cooling to rt the mixture was filtered thougha pad of celite, this was washed with portions of EA. The combinedsolution was concentrated under reduced pressure before being dissolvedin EA (500 mL), washed with brine (150 mL×2) and then dried over sodiumsulfate, filtered and concentrated again. The crude mixture was stirredfor 5 min in chloroform (250 mL) and filtered through a sintered funnel.The filtrate was concentrated to give the crude product as a pale yellowoil. The crude product was purified by CC in (PE/EA=9:1, PE=petroleumether) to give the desired product 1c (2.5 g, 38% for 2 steps) as a paleyellow oil.

Step 3: Methyl3-(3-(2-chloro-4-((5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazol-4-yl)methoxy)phenyl)-3-hydroxycyclobutyl)benzoate(1)

To a stirring solution of compound 1a (1.67 g, 3.5 mmol) in dry THF (30mL) was added n-BuLi (2.5 M in hexane, 1.2 eq, 1.69 mL) dropwise over 10min at −78° C. under a nitrogen atmosphere. This was stirred for 1 h atthis temperature before adding a solution of compound 1c (0.72 g, 1 eq)in dry THF (10 mL) dropwise and stirred for 1 h at this temperature. Thereaction mixture was allowed to warm to rt slowly and left stirringovernight. The reaction was quenched with a solution of saturatedammonium chloride solution (50 mL) and EA (250 mL). The organic layerwas separated and the aq. layer was washed with EA (2×100 mL). Thecombined organic extracts were dried over sodium sulfate, filtered andconcentrated to give the crude product as a brown oil. The product wasisolated following CC with PE/EA (19:1 to 3:1). The reaction andpurification was repeated twice on the same scale and the combinedproduct (3.13 g) was repurified under the same conditions to afford thefinal product 1 (1.7 g, 19%). ¹H NMR (CDCl₃): 7.93 (m, 1H), 7.90-7.85(m, 1H), 7.50-7.30 (m, 5H), 6.88 (s, 1H), 6.75-6.72 (m, 1H), 4.80 (s,2H), 3.88 (s, 3H), 3.20-3.10 (m, 1H), 3.00-2.91 (m, 2H), 2.60-2.49 (m,2H), 2.15-2.08 (m, 1H), 1.30-1.25 (m, 2H), 1.15-1.10 (m, 2H).

Example 2 3-((1s,3s)-3-(2-Chloro-4-((5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazol-4-yl)methoxy)phenyl)-3-hydroxycyclobutyl)benzoicacid (2)

Compound 1 (1.7 g, 2.84 mmol) was dissolved in THF (100 mL) at rt. Asolution of LiOH (285 mg, 4.2 eq) in water (20 mL) was added and thesolution was stirred and warmed to 35° C. for three days. After thistime the THF was removed under reduced pressure. The remaining aq.solution was diluted with water (25 mL) and washed with Et₂O (2×50 mL).The aq. layer was then transferred to a round bottomed flask andacidified to pH 6 using 1N HCl. The formed white precipitated wasfiltered off and dried under reduced pressure at 50° C. to give titlecompound 2 (1.3 g, 78%, single isomer by ¹-NMR and LC-MS) as whitesolid. ¹H NMR (400 MHz, CD₃OD) δ: 7.98 (s, 1H), 7.86 (d, J=7.6 Hz, 1H),7.58-7.46 (m, 5H), 7.41 (t, J=7.6 Hz, 1H), 6.91 (d, J=2.4 Hz, 1H), 6.80(dd, J=8.8, 2.4 Hz, 1H), 4.95 (s, 2H), 3.29-3.25 (m, 2H), 2.96 (m, 1H),2.55-2.49 (m, 2H), 2.37 (m, 1H), 1.24-1.22 (m, 4H). MS (ESI⁻) m/z: 584(582) [M−1]⁻.

Relevant intensive NOEs (obtained from the ROESY spectra; arrows below)indicate that the two aromatic moieties are 1,3-trans oriented inExample 2.

Alternative Route to Example 2 Step 1: 3-(3-Bromophenyl)cyclobutanone(2a)

N,N-Dimethylacetamide (9.0 g, 103 mmol) was dissolved in1,2-dichloroethane (200 mL). The solution was cooled to 0° C. beforetrifluoromethanesulfonic anhydride (63 g, 223 mmol) was added. Thereaction was stirred for an additional 60 min at 0° C. Then1-bromo-3-vinylbenzene (15 g, 81.9 mmol) and 2,4,6-collidine (10.5 g,86.6 mmol) were added. The reaction was heated to reflux overnight,quenched by addition of water (300 mL) and stirred for 2 hr at rt. Themixture was extracted with DCM (300 mL×3). The combined organic layerswere dried over Na₂SO₄ and concentrated in vacuo. Purification by CC(EA/PE=1:20) gave the title compound 2a (5.0 g, 27%) as a pale yellowsolid.

Step 2: 3-(3-Bromophenyl)-1-(2-chloro-4-((5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazol-4-yl)methoxy)phenyl)cyclobutanol (2b)

To a solution of compound la (14 g, 29.6 mmol) in dry THF (500 ml) at−78° C. was added dropwise n-BuLi (18.5 mL, 1.6 M in hexane, 29.6 mmol).The mixture was stirred for an additional 1 h at −78° C. and a solutionof compound 2a (6.5 g, 28.9 mmol) in dry THF (50 mL) was added dropwise.The resulting mixture was stirred at −78° C. for 1 h and then warmed tort and quenched with saturated aq. NH₄Cl (500 mL). The mixture wasextracted with EA (500 mL×2), the combined organic layers were washedwith brine, dried over Na₂SO₄ and concentrated in vacuo. The residue waspurified by CC (EA/PE=1:5) to give the title compound 2b (6.5 g, 37%) asa white solid.

Step 3: 3-(3-Cyanophenyl)-1-(2-chloro-4-((5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazol-4-yl)methoxy)phenyl)cyclobutanol (2c)

To a solution of compound 2b (3.1 g, 5 mmol) in DMF (50 mL) were addedunder argon atmosphere Zn(CN)₂ (500 mg, 4.3 mmol), Pd₂(dba)₃ (300 mg, 0.33 mmol) and Xantphos (150 mg, 0.31 mmol). The mixture was stirred for10 h at 115° C. under microwave irradiation. After cooling to rt thereaction mixture was diluted with water (250 mL) and extracted with EA(250 mL×2). The combined organic layers were washed with brine (100mL×3) and dried over Na₂SO₄. The residue was purified by CC (EA/PE) togive the title compound 2c (1.2 g, 42%) as a pale yellow solid.

Step 4:3-((1s,3s)-3-(2-Chloro-4-((5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazol-4-yl)methoxy)phenyl)-3-hydroxycyclobutyl)benzoicacid (2)

To a solution of compound 2c (15 g, 24.2 mmol) in EtOH (750 mL) wasadded aq. NaOH (40 g in 100 mL of water). The resulting mixture washeated to reflux overnight and then cooled to rt. The reaction wasconcentrated in vacuo to remove the volatile solvent, diluted with water(1000 mL) and the pH was adjusted to 2 with diluted aq. HCl (1 N). Theformed precipitate was collected by filtration to give the crude productas a yellow solid (13.8 g). Purification by preparative preversed phaseHPLC (RP-HPLC) afforded the title compound 2 (8.0 g, 56%, single isomerby ¹H-NMR) as a white solid.

Preparative Example 3

Step 1: Methyl 3-(3-hydroxyazetidin-1-yl)benzoate (3a)

To a solution of methyl 3-iodobenzoate (4.5 g, 17.2 mmol) in DMSO (30mL) was added 3-azetidin-3-ol hydrogen chloride salt (1.3 g, 11.8 mmol),Cs₂CO₃ (9.5 g, 29.2 mmol), CuI (446 mg, 2.3 mmol) and L-proline (540 mg,4.7 mmol) and then the mixture was heated at 90° C. for 18 h under argonatmosphere. The solution was diluted with EA and water and the organiclayer was washed with brine three times, concentrated under reducedpressure and purified by CC (PE/EA=2:1) to give compound 3a (1.6 g, 66%)as a yellow solid.

Step 2: Methyl 3-(3-oxoazetidin-1-yl)benzoate (3)

To a solution of compound 3a (1.60 g, 7.7 mmol) in dry DCM (30 mL) wasadded Dess-Martin periodinane (6.5 g, 15.4 mmol) at 0° C. and themixture was stirred at rt for 2 h under N₂ atmosphere. The mixture wasquenched with saturated sodium bicarbonate solution and diluted with EA.The organic portion was washed with brine, dried over Na₂SO₄, filtered,concentrated under reduced pressure and purified by CC (PE/EA=4:1) togive compound 3 (1.2 g, 75%) as a white solid.

Example 4:3-((1s,3s)-3-(2-Chloro-4-((5-cyclopropyl-3-(2.6-dichlorophenyl)isoxazol-4-yl)methoxy)phenyl)-3-hydroxycyclobutyl)-N-(methylsulfonyl)benzamide(4)

To the solution of compound 2 (100 mg, 0.17 mmol) in DCM (5 mL) wereadded EDCl-HCl (100 mg, 0.52 mmol), DMAP (100 mg, 0.81 mmol) andMeSO₂NH₂ (40 mg, 0.42 mmol). The mixture was stirred at 30° C. overnightand then diluted with EA and washed by H₂O, brine and dried over Na₂SO₄.Concentration in vacuo and purification by prep-TLC gave crude targetcompound as a light yellow solid. RP-HPLC purification afforded thetitle compound 4 (38 mg, 33%) as a white solid. ¹H NMR (400 MHz, CD₃OD)δ: 7.87 (s, 1H), 7.74 (d, J=7.6 Hz, 1H), 7.61-7.53 (m, 4H), 7.50-7.46(m, 2H), 6.91 (d, J=2.4 Hz, 1H), 6.80 (dd, J=8.8, 2.4 Hz, 1H), 4.95 (s,2H), 3.38(s, 3H), 3.30-3.26 (m, 2H), 3.01 (m, 1H), 2.57-2.51 (m, 2H),2.37 (m, 1H), 1.25-1.23 (m, 4H). MS (ESI⁻) m/z: 659 [M−1]⁻.

Example 5 3-(3-(2-Chloro-4-((5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazol-4-yl)methoxy)phenyl)-3-hydroxycyclobutyl)benzenesulfonamide (5)

Step 1: 3-(3-(Benzylthio)phenyl)-1-(2-chloro-4-((5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazol -4-yl)methoxy)phenyl)cyclobutanol (5a)

To a solution of compound 2b (619 mg, 1 mmol) in toluene (20 mL) underargon atmosphere were added K₂CO₃ (276 mg, 2 mmol), phenylmethanethiol(125 mg, 1 mmol), Pd₂(dba)₃ (200 mg, 0. 22 mmol) and Xantphos (75 mg,0.16 mmol). Then the mixture was stirred at 115° C. for 4 h. After beingcooled to rt, the reaction was diluted with water (100 mL) and extractedwith EA (100 mL×2). The combined organic layers were washed with brine(100 mL×2), dried over Na₂SO₄ and concentrated to dryness. Purificationby CC gave compound the compound 5a (200 mg; 30%) as a pale yellowsolid. ¹H NMR (400 MHz, CDCl₃) δ: 7.36-7.32 (m, 3H), 7.28-7.07 (m, 9H),7.01 (d, J=7.2 Hz, 1H), 6.82 (d, J=2.0 Hz, 1H), 6.66 (dd, J=8.8, 2.0 Hz,1H), 4.75 (s, 2H), 4.04 (s, 2H), 3.06-3.00 (m, 2H), 2.84-2.78 (m, 2H),2.44-2.38 (m, 3H), 2.09 (m, 1H), 1.24-1.18 (m, 2H), 1.11-1.08 (m, 2H).MS (ESI⁺) m/z: 662 [M+1]⁺.

Step 2:3-(3-(2-Chloro-4-((5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazol-4-yl)methoxy)phenyl)-3-hydroxycyclobutyl)benzene-1-sulfonylchloride (5b)

To a solution of compound 5a (34 mg, 0.05 mmol) in CH₃CN/HOAc/H₂O (1mL/37 μL/25 μL) was added 2,4-dichloro-5,5-dimethylhydantion (20 mg, 0.1mmol). The mixture was stirred at 0-5° C. for 2 h. The reaction wasdiluted with water and extracted with CH₂Cl₂. The combined organiclayers were washed with a 5% NaHCO₃ solution, brine and dried overNa₂SO₄. Concentration to dryness afforded the crude product 5b (30 mg)as a colorless oil, which was used directly in the next step.

Step 3: 3-(3-(2-Chloro-4-((5-cyclopropyl-3-(26-dichlorophenyl)isoxazol-4-yl)methoxy)phenyl)-3-hydroxycyclobutyl)benzenesulfonamide(5)

To the solution of compound 5b (30 mg) in CH₃CN (2 mL) was added NH₄OH(0.3 mL). The mixture was stirred at rt for 1 h. Concentration todryness and purification by prep. RP-HPLC gave the title compound 5 (3.5mg, 10% for two steps) as a white solid. ¹H NMR (400 MHz, CDCl₃) δ: 7.85(s, 1H), 7.77 (d, J=7.6 Hz, 1H), 7.54-7.41 (m, 5H), 7.35 (d, J=8.4 Hz,1H), 6.90 (s, 1H), 6.75 (d, J=8.4 Hz, 1H), 4.83 (s, 2H), 4.77 (s, broad,2H), 3.20 (t, J=10.4 Hz, 2H), 3.04 (m, 1H), 2.58 (t, J=10.6 Hz, 2H),2.17 (m, 1H), 1.31-1.30 (m, 2H), 1.20-1.16 (m, 2H). MS (ESI⁻) m/z: 617[M−1]⁻.

Example 61-(2-Chloro-4-((5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazol-4-yl)methoxy)phenyl)-3-(3-(methylsulfonyl)phenyl)cyclobutanol (6)

To the solution of compound 2b (200 mg, 0.32 mmol) in DMSO, sodiummethanesulfinate (50 mg, 0.46 mmol), CuI (20 mg, 0.1 mmol), L-proline(37 mg, 0.32 mmol) and diisopropylethylamine (DIEA) (41 mg, 0.32 mmol)was added. The mixture was stirred at 95° C. overnight and then dilutedwith water and extracted with EA. The combined organic layers werewashed with water and dried over Na₂SO₄. Concentration to dryness underreduced pressure and purification by prep. RP-HPLC gave the titlecompound 6 as a white solid (35 mg, 21%, single isomer by ¹H NMR andLC-MS). ¹H NMR (400 MHz, CDCl₃) δ: 7.84 (s, 1H), 7.79 (d, J=7.6 Hz, 1H),7.60 (d, J=7.6 Hz, 1H), 7.53 (t, J=7.6 Hz, 1H), 7.44-7.41 (m, 3H), 7.34(t, J=7.2 Hz, 1H), 6.90 (d, J=2.8 Hz, 1H), 6.75 (dd, J=8.4, 2.0 Hz, 1H),4.83 (s, 2H), 3.24-3.19 (m, 2H), 3.08-3.04 (m, 4H), 2.62-2.56 (m, 2H),2.17 (m, 1H), 1.31-1.29 (m, 2H), 1.20-1.16 (m, 2H). MS (ESI+) m/z: 618(620) [M+1]⁺, 600 (602) [M−H₂O+1 ]⁺.

Example 7 Methyl5-((1s,3s)-3-(2-chloro-4-((5-cycloproyl-3-(2,6-dichlorophenyl)isoxazol-4-yl)methoxy)phenyl)-3-hydroxycyclobutyl)-1-isopropyl-1H-pyrazole-3-carboxylate(7)

Step 1: Methyl 1-isopropyl-5-vinyl-1H-pyrazole-3-carboxylate (7a)

A suspension of methyltriphenylphosphonium bromide (2.69 g, 7.52 mmol)in dry THF (40 mL) was cooled to −78° C. and n-butyllithium (1.6 Msolution in hexane, 3.7 mL, 5.91 mmol) was added dropwise. Theyellow-orange suspension was stirred at −78° C. for 50 min and then asolution of methyl 5-formyl-1-isopropyl-1H-pyrazole-3-carboxylate(prepared as described in WO 2011/020615, 1.05 g, 5.37 mmol) in dry THF(10 mL) was added dropwise. The mixture was stirred at −78° C. for 1.75h, the cooling bath was removed and the mixture (off-white suspension)was stirred at rt for 1 h. The mixture was then partitioned betweendiluted aq. NaHCO₃ solution (150 mL) and EA (150 mL). The aq. layer wasextracted twice with EA (50 mL each) and the combined organic layer waswashed twice with water (50 mL each) and concentrated without drying togive 2.74 g of a yellow oil which slowly crystallized. The crude productwas purified by CC (preadsorption with CH₂Cl₂, hexane/EA 4:1) to givealkene 7a (590 mg, 57%) as a colorless oil. ¹H NMR (DMSO-d₆) δ: 7.02 (s,1H), 6.87 (dd, J=17.3, 11.2 Hz, 1H), 5.94 (dd, J=17.3, 1.3 Hz, 1H), 5.45(dd, J=11.2, 1.3 Hz, 1H), 4.80 (sept, J=6.6 Hz, 1H), 3.79 (s, 3H), 1.38(d, J =6.6 Hz, 6H). C₁₀H₁₄N₂O₂ (194.23). LC-MS (ESI): 195 [M₊H]⁺.

Step 2: Methyl 1-isopropyl-5-(3-oxocyclobutyl)-1H-pyrazole-3-carboxylate(7b)

The reaction was performed in two dry sealed tubes (two batches of equalquantity). The batches were combined for workup and purification. Singlebatch procedure: To a solution of N,N-dimethylacetamide (0.22 mL, 2.34mmol) in 1,2-dichloroethane (12 mL) under nitrogen at −15 to −20° C. wasadded dropwise trifluoromethanesulfonic anhydride (0.43 mL, 2.57 mmol),forming an opaque suspension. The mixture was stirred at −15° C. for 10min, and a solution of alkene 7a (151 mg, 0.78 mmol) and sym.-collidine(0.42 mL, 3.12 mmol) in 1,2-dichloroethane (3 mL) was added dropwise(yellow solution formed). Upon completion of the addition the coolingwas bath removed, the mixture was allowed to warm to rt (orange turbidsolution) and the tube was sealed. The mixture was then stirred at 90°C. for 15 h (brown mixtures). Water (5 mL) was added at rt and themixtures were stirred at 100° C. for 2 h (turbid two-phase solutions).After cooling to rt, the mixtures were combined and partitioned betweendiluted aq. NaHCO₃ solution and CH₂Cl₂ and the aq. layer was extractedthree times with CH₂Cl₂ (30 mL each). The combined organic layer wasdried (Na₂SO₄), filtered and concentrated to give a brown oil (2.2 g).Purification by CC (6×13 cm, preadsorption with CH₂Cl₂, toluene/EA 3:1)gave cyclobutanone 7b (115.5 mg, 31%) as a yellow oil. ¹H NMR (DMSO-d₆)δ: 6.81 (s, 1H), 4.58 (sept, J=6.5 Hz, 1H), 3.78 (s, 3H), 3.85-3.73 (m,1H), 3.59-3.45 (m, 2H), 3.37-3.24 (m, 2H, partially overlapped by watersignal), 1.39 (d, J=6.6 Hz, 6H). C₁₂H₁₆N₂O₃ (236.27). LC-MS (ESI): 237[M+H]⁺.

Step 3: Methyl5-((1s,3s)-3-(2-chloro-4-((5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazol-4-yl)methoxy)phenyl)-3-hydroxycyclobutyl)-1-isopropyl-1H-pyrazole-3-carboxylate(7)

A solution of bromide 1a (368 mg, 0.78 mmol) in dry THF (6 mL) wascooled to −78° C. and a 1.6M n-butyllithium solution in hexanes (0.48mL, 0.76 mmol) was added dropwise. The mixture was stirred at −78° C.for 20 min and a solution of cyclobutanone 7b (164 mg, 0.69 mmol) in dryTHF (4 mL) was added dropwise. The mixture was stirred at −78° C. for2.5 h and saturated aq. NH₄Cl solution (1 mL) was added dropwise at thistemperature. The cooling bath was removed and the mixture was allowed towarm to rt and stirred at rt for 0.5 h. The mixture was then added todiluted aq. NH₄Cl solution and extracted three times with EA. Thecombined organic layer was dried (Na₂SO₄), filtered and concentrated togive 516 mg of an almost colorless oil. Purification by CC (4.5×23 cm,preadsorption with CH₂Cl₂, eluent hexane/acetone=2:1) afforded recoveredcyclobutanone 7b (31.3 mg, 19%, slightly yellow oil) and impure product(333 mg). Repurification by CC (4×22 cm, hexane/EA=1:1) or prep-TLC gavepure product 7 (210 mg, 48%) as white foam. ¹H NMR (DMSO-d₆) δ: 7.65 (d,J=2.1 Hz, 1H), 7.62 (s, 1H), 7.59-7.48 (m, 2H), 6.92 (d, J=2.4 Hz, 1H),6.76 (dd, J =8.6, 2.6 Hz, 1H), 6.66 (s, 1H), 5.49 (s, 1H), 4.92 (s, 2H),4.42 (quint-like m, J=6.5 Hz, 1H), 3.78 (s, 3H), 3.24-3.11 (m, 2H,partially overlapped by water signal), 3.04-2.90 (m, 1H), 2.54-2.33 (m,3H, partially overlapped by DMSO signal), 1.32 (d, J=6.5 Hz, 6H),1.26-1.08 (m, 4H). C₃₁H₃₀Cl₃N₃O₅ (630.95). LC-MS (ESI): 630, 632 [M+H]⁺.

Example 85-((1s,3s)-3-Chloro-4-((5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazol-4-yl)methoxy)phenyl)-3-hydroxycyclobutyl)-1-isopropyl-1H-pyrazole-3-carboxylicacid (8)

Ester 7 (98.3 mg, 0.156 mmol) was dissolved in a mixture of THF (7.5mL), MeOH (2.5 mL) and water (2.5 mL) and LiOH.H₂O (65 mg, 1.56 mmol)was added at rt. The mixture was stirred at rt for 18 h. The mixture waspartitioned between diluted aq. NH₄Cl solution and EA and the organiclayer was washed once with water. The combined aq. layer was extractedtwice with EA. The combined organic layer was dried (Na₂SO₄), filteredand concentrated to give 103 mg of an almost white solid. The productwas purified by CC (3×3.5 cm, EA/EtOH=10:1 to 1:4) to afford 8 (94.8 mg,99%) as a white solid. ¹H NMR (DMSO-d₆) δ: 7.66-7.60 (m, 1H), 7.62 (s,1H), 7.59-7.49 (m, 2H), 6.91 (d, J=2.5 Hz, 1H), 6.76 (dd, J=8.6, 2.4 Hz,1H), 6.38 (s, 1H), 5.51 (s, 1H, exchangeable with D₂O), 4.92 (s, 2H),4.31 (quint-like m, J=6.5 Hz, 1H), 3.25-3.08 (m, 2H, partiallyoverlapped by water signal), 2.93-2.77 (m, 1H), 2.57-2.43 (m, 1H, hiddenby DMSO signal), 2.43-2.29 (m, 2H, partially overlapped by DMSO signal),1.29 (d, J=6.5 Hz, 6H), 1.26-1.08 (m, 4H). The CO₂H signal does notappear in the spectrum. C₃₀H₂₈Cl₃N₃O₅ (616.92). LC-MS (ESI): 616, 618[M+H]⁺.

Alternative route to Example 8 Step 1: 1-(3-Methylenecyclobutyl)ethanone(8a)

Methylene cyclobutane carbonitrile (5.0 g, 53.7 mmol) was dissolved indry diethylether (25 mL), cooled in an ice bath and MeMgBr (26.8 mL,80.5 mmol, 3 M in ether) was added dropwise. The mixture was leftstirring overnight at rt, cooled to 0° C., quenched carefully with 15%NaHSO₄ aq. sol. (100 mL). The mixture was stirred at rt for 30 min. andthe layers were separated. The aq. phase was extracted with pentane (50mL) and diethylether (50 mL). The combined organic layers were washedwith brine and dried over Na₂SO₄. The solvents were removed under vacuumat rt and the crude product was obtained as a yellowish liquid.

Step 2: Ethyl 4-(3-methylenecyclobutyl)-2,4-dioxobutanoate (8b)

Sodium (1.15 g, 49.9 mmol) was dissolved in dry EtOH (30 mL, denaturatedwith 5% diethylether). Compound 8a (5.5 g, 49.9 mmol, crude) wasdissolved in dry EtOH (45 mL) and the above prepared sodium ethoxidesolution was added. This mixture was stirred at rt for 15 min and thendiethyl oxalate (6.8 mL, 49.9 mmol) was added dropwise. The reactionmixture was placed in a pre-heated (to 67 ° C.) oil bath and stirred atthis temperature for 4.5 h. The mixture was left at rt overnight. Thesolvent was removed, EA (100 mL) and 1 M HCl (70 mL) were added andorganic phase was separated. The aq. phase was re-extracted with EA (50mL). The combined organic phases were washed with water, brine and driedover anh. Na₂SO₄. The solvent was removed under reduced pressure and theresidue was purified on silica using hexanes/MTBE 9:1 as eluent givingpure product 8b. Yield: 6.29 g, 56% over two steps. ¹H-NMR (CDCl₃), δ(ppm): 6.36 (s, 1H), 4.85-4.80 (m, 2H), 4.34 (q, J=8.0 Hz, 2H),3.35-3.25 (m, 1H), 3.05-2.85 (m, 4H), 1.36 (t, J=8.0 Hz, 3H).

Step 3: Ethyl1-isopropyl-5-(3-methylenecyclobutyl)-1H-pyrazole-3-carboxylate (8c)

Compound 8b (6.29 g, 29.9 mmol) was dissolved in dry EtOH (65 mL,denaturated with 5% of MeOH) and isopropyl hydrazine hydrochloride (3.97g, 35.9 mmol) was added. The reaction mixture was stirred for 3 h at rt.The solvent was removed and to the oily residue were added EA (100 mL),water (50 mL) and sat. NaHCO₃ (50 mL) sequentially. The layers wereseparated and the aq. phase was re-extracted with EA (50 mL). Thecombined organic phases were washed with brine (70 mL) and dried overanh. Na₂SO₄. The solvent was removed under vacuum and the residue wasdried under reduced pressure. Yield: 7.23 g (contains 3.4% of EtOAc byNMR, recalculated pure yield: 6.98 g, 94%). Crude product 8c is 98% pureby HPLC and NMR. ¹H-NMR (CDCl₃), δ (ppm): 6.62 (s, 1H), 4.88-4.82 (m,2H), 4.42-4.32 (m, 3H), 3.56-3.45 (m, 1H), 3.17-3.07 (m, 2H), 2.88-2.79(m, 2H), 1.49 (d, J=8.0 Hz, 6H), 1.37 (t, J=8.0 Hz, 3H).

Step 4: Ethyl 1-isopropyl-5-(3-oxocyclobutyl)-1H-pyrazole-3-carboxylate(8d)

Compound 8c (6.45 g, 26.0 mmol) was dissolved in a mixture of MeCN (77mL) and water (13 mL) and cooled in an ice-bath. To this solutionRuCl₃×H2O (0.19 g, 0.86 mmol) was added, followed by portion-wiseaddition of NaIO₄ (19.35 g, 90.9 mmol). An exotherm was observed duringthis addition. The obtained thick slurry was stirred at rt for 45 min.The reaction mixture was diluted with Na₂S₂O₃ aq. sol. (10%, 260 mL),water (50 mL) and DCM (100 mL). The phases were separated and the aq.phase was extracted with DCM (2×70 mL). The combined organic phases werewashed with Na₂S₂O₃ aq. sol. (10%, 50 mL), water (100 mL), brine (100mL) and dried over anh. Na₂SO₄. The crude product (6.5 g) was purifiedon silica, eluting with hexanes/MTBE to give pure product as an oil thatsolidified upon storage at −20° C. Yield: 5.8 g (78% over two steps).¹H-NMR (DMSO-d₆), δ (ppm): 6.78 (s, 1H), 4.57 (h, J=8.0 Hz, 1H), 4.26(q, J=8.0 Hz, 2H), 3.85-3.75 (m, 1H), 3.58-3.45 (m, 2H), 3.35-3.25 (m,2H), 1.39 (d, J=8.0 Hz, 6H), 1.28 (t, J=8.0 Hz, 3H).

Step 5:4-((4-Bromo-3-chlorophenoxy)methyl)-5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazole(8e)

3-Chloro-4-bromophenol (3.8 g, 18.3 mmol) was mixed with(5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazol-4-yl)methanol (3.47 g,12.2 mmol) and triphenylphosphine (6.41 g, 24.4 mmol) in toluene (150mL). The mixture was cooled in an ice-bath and DIAD (4.8 mL, 24.4 mmol)as a solution in toluene (10 mL) was added drop-wise. The reaction wasstirred at rt for 21 h and the solvents were removed on a rotavapleaving a yellow oily residue. This was dissolved in DCM (200 mL),silica (˜20 g) was added and the mixture was evaporated to dryness. Thismaterial was loaded on the top of a silica column and purified elutingwith hexanes/MTBE 9:1. The product containing fractions were pooled andthe solvent removed under reduced pressure, leaving pure product 8e as acolourless oil that crystallized upon drying under vacuum overnight.Yield: 5.07 g (88%).¹H-NMR (CDCl₃), δ (ppm): 7.45-7.30 (m, 4H), 6.90 (s,1H), 6.60-6.55 (m, 1H), 2.15-2.07 (m, 1H), 1.32-1.25 (m, 2H), 1.20-1.11(m, 2H).

Step 6: Ethyl5-((1s,3s)-3-(2-chloro-4-((5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazol-4-yl)methoxy)phenyl)-3-hydroxycyclobutyl)-1-isopropyl-1H-pyrazole-3-carboxylate(8f)

LiCl (0.684 g, 16.15 mmol) was dissolved in THF (20 mL) at rt andiPrMgCl (2.0 M in THF, 8.1 mL, 16.15 mmol) was added. The mixture wasstirred for 10 min at rt, cooled in an ice-bath and a solution ofcompound 8e (2.55 g, 5.38 mmol) in THF (20 mL) was added over 5 min. Thecooling bath was removed and the mixture was stirred at rt for 4 h. Themixture was cooled to −10° C. and a solution of compound 8d (1.48 g,5.92 mmol) in THF (16 mL) was added rapidly. The mixture was stirred atrt for 90 min. and then 0.5 M NaHSO₄ aq. (35 mL) and EA (50 mL) wereadded. The resulting mixture was stirred for 10 min., the layers wereseparated and the aq. layer was extracted with EA (30 mL). The combinedorganic phases were washed with NaHCO₃ aq. (50 mL), brine (50 mL) anddried over anh. Na₂SO₄. The crude product (3.79 g) was obtained afterremoval of the solvent as a white foam. 3.6 g of this crude was purifiedon silica column, eluting with hexanes/EA 3:2 to give pure product 8f asa solid foam. Yield: 1.62 g (49%). ‘H-NMR (DMSO-d₆), δ (ppm): 7.65-7.47(m, 4H), 6.93-6.91 (m, 1H), 6.79-6.72 (m, 1H), 6.65 (s, 1H), 5.48 (s,1H), 4.92 (s, 2H), 4.42 (h, J=8.0 Hz, 1H), 4.26 (q, J=8.0 Hz, 2H), 3.32(s, 2H), 3.22-3.14 (m, 2H), 3.05-2.90 (m, 1H), 2.45-2.35 (m, 2H),1.35-1.10 (m, 14H).

Step 7:5-((1s,3s)-3-(2-Chloro-4-((5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazol-4-yl)methoxy)phenyl)-3-hydroxycyclobutyl)-1-isopropyl-1H-pyrazole-3-carboxylic acid (8)

Compound 8f (1.60 g, 2.48 mmol) was dissolved in THF (100 mL), then MeOH(50 mL), water (50 mL) and LiOH×H2O (1.04 g, 24.8 mmol) were addedsequentially. The mixture was stirred for 4.5 h at rt and thenconcentrated under reduced pressure to remove MeOH and THF. Theremaining aq. solution was acidified by addition of 1 M HCl aq. (24 mL)to reach pH of 4.05 (pH electrode control). Already at approx. pH 7 aprecipitate started to form. The formed solid was filtered off, washedon the filter with water and dried under vacuum at rt to give product 8as a white powder. Yield: 1.40 g (92%). ¹H-NMR (CDCI₃), δ (ppm):7.44-7.32 (m, 4H), 6.91 (d, J=4.0 Hz, 1H), 6.78 (s, 1H), 6.75 (dd, J=4.0Hz, J=8.0 Hz, 1H), 4.83 (s, 2H), 4.35-4.20 (m, 1H), 3.25-3.14 (m, 2H),3.04-2.90 (m, 1H), 2.62-2.54 (m, 2H), 2.21-2.11 (m, 1H), 1.46 (d, J=8.0Hz, 6H), 1.34-1.28 (m, 2H), 1.20-1.14 (m, 2H). ³C-NMR (CDCl₃), δ (ppm):172.7, 164.8, 159.2, 158.4, 147.2, 141.3, 135.8, 134.1, 132.8, 131.3,128.1, 127.6, 127.3, 117.7, 113.3, 110.0, 106.3, 73.1, 59.8, 51.1, 41.7,22.6, 22.0, 8.5, 7.8. MS (ESI⁺) m/z: 616.4 [M+1]⁺.

Example 8A5-((1r,3r)-3-(2-Chloro-4-((5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazol-4-yl)methoxy)phenyl)-3-hydroxycyclobutyl)-1-isopropyl-1H-pyrazole-3-carboxylate(8A)

Example 8A can be prepared by subjecting the crude product 8f to theester hydrolysis as described for 8 and isolation from the crude product8 as a minor isomer by preparative RP-HPLC. ¹H-NMR (CDCl₃), δ (ppm):7.42-7.30 (m, 2H), 7.11 (d, J=8.0 Hz, 1H), 6.75-6.65 (m, 1H), 6.57 (s,1H), 4.79 (s, 2H), 4.50-4.41 (m, 1H), 3.96-3.85 (m, 1H), 2.98-2.90 (m,2H), 2.67-2.57 (m, 2H), 2.20-2.09 (m, 1H), 1.51 (d, J=8.0 Hz, 6H),1.32-1.14 (m, 4H). ³C-NMR (CDCl₃), δ (ppm): 172.6, 166.2, 159.2, 158.4,147.4, 141.2, 135.7, 134.6, 132.8, 131.3, 128.1, 127.7, 127.5, 116.8,113.5, 110.0, 105.8, 75.1, 59.8, 51.2, 41.8, 25.4, 22.6, 8.5, 7.8. MS(ESI⁺) m/z: 616.3 [M+1]⁺.

The transannular configuration of the major isomer (compound 8) and theminor isomer (compound 8A) was confirmed by NOE experiments. Thedetected indicative NOEs between protons are indicated in the followingpictures by double arrows:

NOEs Detected for Eexample 8with 1,3-Ttrans Transannular Configurationof the Aromatic Moieties

NOEs Detected for Example 8A with 1,3-cis Transannular Configuration ofthe Aromatic Moieties Example 9 Methyl6-(3-(2-chloro-4-((5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazol-4-yl)methoxy)phenyl)-3-hydroxycyclobutyl)-1-methyl-1H-indazole-3-carboxylate(9)

Step 1: Methyl 1-methyl -6-vinyl-1H-indazole-3-carboxylate (9a)

To the solution of methyl 6-bromo-1-methyl-1H-indazole-3-carboxylate (60mg, 0.22 mmol) in DMF (10 mL), tributyl(vinyl)tin (99 μL, 0.34 mmol),Pd(Ph₃)₄ (11 mg, 9 μmol) was added. After the addition was completed,the mixture was stirred at 90° C. for 4 h under Ar. Then the solvent wasremoved under reduced pressure. Purification by CC afforded compound 9a(52 mg, 88%).

Step 2: Methyl 1-methyl-6-(3-oxocyclobutyl)-1H-indazole-3-carboxylate(9b)

Following the procedure as described in Example 7/Step 2, compound 9bwas obtained from 9a in 57% yield. ¹H NMR (400 MHz, CDCl₃) δ: 8.14 (d,J=8.4 Hz, 1H), 7.31 (s, 1H), 7.23 (d, J=8.8 Hz, 1H), 4.13 (s, 3H), 3.99(s, 3H), 3.87-3.79 (m, 1H), 3.58-3.51 (m, 2H), 3.33-3.26 (m, 2H). m/z:259 [M+1]⁺.

Step 3: Methyl 6-(3-(2-chloro-4-((5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazol-4-yl)methoxy)phenyl)-3-hydroxycyclobutyl)-1-methyl-1H-indazole-3-carboxylate(9)

Following the procedure as described in Example 7/Step 3, compound 9 wasobtained from 9b in 40% yield.

Example 106-(3-(2-Chloro-4-((5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazol-4-yl)methoxy)phenyl)-3-hydroxycyclobutyl)-1-methyl-1H-indazole-3-carboxylicacid (10)

Following the procedure as described in Example 8, compound 10 wasobtained from compound 9 in 45% yield as a white solid. ¹H NMR (400 MHz,CDCl₃) δ: 8.14 (d, J=8.0 Hz, 1H), 7.48 (d, J=8.8 Hz, 1H), 7.43-7.32 (m,4H), 7.29 (m, 1H), 6.92 (d, J=2.4 Hz, 1H), 6.76 (dd, J=7.2 Hz, 2.4 Hz,1H), 4.84 (s, 2H), 4.18 (s, 3H), 3.45-3.40 (m, 1H), 3.28-3.23 (m, 2H),3.19-3.10 (m, 1H), 2.68-2.63 (m, 2H), 2.21-2.14 (m, 1H), 1.33-1.29 (m,2H), 1.20-1.15 (m, 2H). m/z: 638 [M+1]⁺.

Preparative Example 11

Step1: Methyl 5-(3-hydroxyazetidin-1-yl)nicotinate (11 a)

A mixture of methyl 5-bromonicotinate (2.00 g, 9.26 mmol), azetidin-3-ol(1.01 g, 9.26 mmol), Cs₂CO₃ (9.06 g, 27.8 mmol), BINAP (1.15 g, 1.85mmol) and Pd(OAc)₂ (0.44 g, 1.85 mmol) in dry dioxane (115 mL) washeated overnight at 85° C. under N₂ atmosphere. The resulting mixturewas filtrated, concentrated under reduced pressure and purified byprep-HPLC to give compound 11a (250 mg, 13%) of as a yellow solid.

Step 2: Methyl 5-(3-oxoazetidin-1-yl)nicotinate (11)

To a solution of compound 11a (250 mg, 1.20 mmol) in dry DCM (15 mL) wasadded Dess-Martin periodinane (1.014 g, 2.40 mmol) at 0° C. under N₂atmosphere and the solution was stirred at rt for 2 h. The resultingsolution was quenched with saturated sodium bicarbonate solution anddiluted with EA. The organic portion was washed with brine, dried overNa₂SO₄, filtered, concentrated under reduced pressure and purified by CC(DCM/MeOH=150:1) to give compound 11 (140 mg, 57%) of as a yellow solid.

Preparative Example 12

Using a similar procedure as that described in Preparative Example 11the following compound has been prepared:

Example 13/1 to 13/9

The following table lists further examples prepared according the abovementioned preparative examples and examples. All listed compounds wereprepared as single isomers.

# Structure Analytical data 13/1

¹H NMR (400 MHz, DMSO-d₆) δ: 1.15-1.25 (m, 4H), 2.35-2.50 (m, 5H,partially under solvent signal), 2.80-2.91 (m, 1H), 3.11-3.20 (m, 2H),4.93 (s, 2H), 6.72-6.81 (m, 1H), 6.93 (s, 1H), 7.40-7.51 (m, 2H),7.52-7.60 (m, 2H), 7.62-7.66 (m, 2H), 7.85-7.90 (m, 2H). MS Calcd.: 583;MS Found: 584 [M + H]⁺. 13/2

¹H NMR (400 MHz, CD₃OD) δ: 1.05-1.12 (m, 4H), 2.18-2.34 (m, 9H),2.62-2.71 (m, 1H), 2.99-3.09 (m, 2H), 4.78 (s, 2H), 6.60-6.64 (m, 1H),6.72-6.78 (m, 1H), 6.85 (s, 2H), 7.30-7.42 (m, 4H). MS Calcd.: 611; MSFound: 612 [M + H]⁺. 13/3

¹H NMR (400 MHz, CD₃OD) δ: 1.10-1.23 (m, 4H), 2.36-2.49 (m, 3H),3.00-3.12 (m, 1H), 3.15-3.25 (m, 2H), 3.87 (s, 3H), 4.95 (s, 2H),6.72-6.80 (m, 1H), 6.89 (s, 1H), 6.90-7.00 (m, 1H), 7.42-7.60 (m, 4H),7.89-7.93 (m, 1H), 7.98 (s, 1H). MS Calcd.: 613; MS Found: 612 [M − H]⁻.13/4

¹H NMR (400 MHz, CD₃OD) δ: 1.20-1.30 (m, 4H), 2.27 (s, 3H), 2.30-2.55(m, 3H), 2.98-3.10 (m, 1H), 3.25-3.40 (m, 2H, partially under solventsignal), 4.95 (s, 2H), 6.76-6.84 (m, 1H), 6.91 (s, 1H), 7.20- 7.25 (m,1H), 7.43-7.63 (m, 4H), 7.75-7.82 (m, 1H), 8.03 (s, 1H). MS Calcd.: 597;MS Found: 596 [M − H]⁻ 13/5

¹H NMR (400 MHz, CD₃OD) δ: 1.20-1.25 (m, 4H), 2.33-2.43 (m, 4H),2.46-2.56 (m, 2H), 2.88-2.97 (m, 1H) 3.22-3.30 (m, 2H), 4.94 (s, 2H),6.78-6.82 (m, 1H), 6.90 (s, 1H), 7.37 (s, 1H), 7.43-7.60 (m, 4H), 7.69(s, 1H), 7.78 (s, 1H). MS Calcd.: 597; MS Found: 596 [M − H]⁻. 13/6

¹H NMR (400 MHz, CD₃OD) δ: 1.17-1.23 (m, 4H), 2.31-2.40 (m, 4H),2.42-2.50 (m, 2H), 2.83-2.92 (m, 1H) 3.19-3.26 (m, 2H), 4.92 (s, 2H),6.75-6.80 (m, 1H), 6.88 (s, 1H), 7.19-7.22 (m, 1H), 7.43-7.57 (m, 4H),7.83 (s, 1H). MS Calcd.: 597; MS Found: 598 [M + H]⁺. 13/7

¹H NMR (400 MHz, CD₃OD) δ: 1.09-1.11 (m, 4H), 2.19-2.26 (m, 1H),4.08-4.10 (m, 2H), 4.19-4.21 (m, 2H), 4.80 (s, 2H), 6.64-6.67 (m, 2H),6.75 (s, 1H), 7.16-7.21 (m, 2H), 7.28-7.39 (m, 6H); MS Calcd.: 584; MSFound: 585 (M + 1). 13/8

¹H NMR (300 MHz, CDCl₃) δ: 1.11 (m, 2H), 1.24 (m, 2H), 2.11 (m, 1H),4.33 (m, 2H), 4.46 (m, 2H), 4.78 (s, 2H), 6.67 (dd, J = 1.2 Hz, 8.4 Hz,1H), 6.77 (d, J = 2.4 Hz, 1H), 6.67 (d, J = 8.4 Hz, 1H), 7.28-7.35 (m,3H), 7.55 (d, J = 1.2 Hz, 1H), 7.94 (s, 1H), 8.56 (d, J = 3.6 Hz, 1H);MS Calcd.: 585; MS Found: 586 (M + 1). 13/9

¹H NMR (300 MHz, DMSO-d₆) δ: 1.13-1.23 (m, 4H), 2.50 (m, 1H), 4.23 (d, J= 8.4 Hz, 2H), 4.51 (d, J = 9.3 Hz, 2H), 4.96 (s, 2H), 6.24 (s, 1H),6.80 (d, J = 7.5 Hz, 1H), 6.88 (s, 1H), 6.97 (s, 1H), 7.07 (s, 1H), 7.44(d, J = 8.4 Hz, 1H), 7.58-7.66 (m, 3H), 8.25 (s, 1H); MS Calcd.: 585; MSFound: 586 (M + 1).

Example 14/1 and 14/2

Using a similar procedure as described in the Examples 1 to 13 andSchemes above, the following compounds were obtained by using theappropriate building blocks.

# Structure Analytical data 14/1

¹H NMR (400 MHz, DMSO-d₆): δ 1.13-1.23 (m, 4H), 1.33 (d, J = 6.4 Hz,6H), 2.37-2.47 (m, 3H), 2.90- 2.95 (m, 1H), 3.14-3.19 (t, J = 8.8 Hz,2H), 3.57 (d, J = 4.0 Hz, 2H), 4.38 (m, 1H), 4.92 (s, 2H), 5.51 (s, 1H),6.51 (s, 1H), 6.76 (dd, J = 2.4 Hz, J = 8.4 Hz, 1H), 6.91 (d, J = 2.4Hz, 1H), 7.51-7.58 (m, 2H), 7.62-7.65 (m, 2H), 7.69 (s, 1H); MS Calcd.:672; MS Found: 673 [M + H]⁺. 14/2

¹H NMR (400 MHz, DMSO-d₆): δ 1.13-1.20 (m, 4H), 1.31 (d, J = 6.0 Hz,6H), 2.36-2.47 (m, 3H), 2.59- 2.63 (m, 2H), 2.89-2.93 (m, 1H), 3.17-3.19(m, 2H), 3.47-3.52 (m, 2H), 4.33-4.39 (m, 1H), 4.92 (s, 2H), 5.51 (s,1H), 6.50 (s, 1H), 6.76 (dd, J = 2.4 Hz, J = 8.4 Hz, 1H), 6.91 (d, J =2.4 Hz, 1H), 7.51-7.58 (m, 2H), 7.62-7.65 (m, 2H), 8.18-8.20 (m, 1H); MSCalcd.: 744; MS Found: 721 [M − Na]⁻. 14/3

¹H NMR (400 MHz, DMSO-d₆): δ 1.10-1.25 (m, 4H), 1.34 (d, J = 6.4 Hz,6H), 3.03-2.95 (m, 1H), 2.50- 2.30 (m, 3H), 3.20-3.10 (m, 2H), 4.50-4.35(m, 1H), 4.92 (s, 2H), 5.5 (s, 1H), 6.92 (s, 1H), 6.78-6.70 (m, 2H),7.70-7.49 (m, 4H), 11.44 (s, 1H); MS Calcd.: 694; MS Found: 695 [M +H]⁺.

The following compound can be prepared in the same manner by usingsimilar procedures as described above:

Assays

FRET Activity Assay

Determination of a ligand mediated cofactor peptide interaction toquantify ligand binding to the nuclear receptor FXR was performed asfollows: Preparation of human FXR alpha ligand binding domain: The humanFXRalpha LBD was expressed in E. coli strain BL21(DE3) as anN-terminally GST tagged fusion protein. The DNA encoding the FXR ligandbinding domain was cloned into vector pDEST15 (Invitrogen). Expressionwas under control of an IPTG inducible T7 promoter. The amino acidboundaries of the ligand binding domain were amino acids 187-472 ofDatabase entry NM_(—)005123 (RefSeq). Expression and purification of theFXR-LBD: An overnight preculture of a transformed E. coli strain wasdiluted 1:20 in LB-Ampicillin medium and grown at 30° C. to an opticaldensity of OD₆₀₀=0.4-0.6. Gene expression was then induced by additionof 0.5 mM IPTG. Cells were incubated an additional 6 h at 30° C., 180rpm. Cells were collected by centrifugation (7000×g, 7 min, rt). Perliter of original cell culture, cells were resuspended in 10 mL lysisbuffer (50 mM Glucose, 50 mM Tris pH 7.9, 1 mM EDTA and 4 mg/mLlysozyme) and left on ice for 30 min. Cells were then subjected tosonication and cell debris removed via centrifugation (22000×g, 30 min,4° C.). Per 10 mL of supernatant 0.5 mL prewashed Glutathione 4Bsepharose slurry (Qiagen) was added and the suspension kept slowlyrotating for 1 h at 4° C. Glutathione 4B sepharose beads were pelletedby centrifugation (2000×g, 15 sec, 4° C.) and washed twice in washbuffer (25 mM Tris, 50 mM KCl, 4 mM MgCl₂ and 1M NaCl). The pellet wasresuspended in 3 mL elution buffer per liter of original culture(elution buffer: 20 mM Tris, 60 mM KCl, 5 mM MgCl₂ and 80 mM glutathioneadded immediately prior to use as powder). The suspension was leftrotating for 15 min at 4° C., the beads pelleted and eluted again withhalf the volume of elution buffer than the first time. The eluates werepooled and dialysed overnight in 20 mM Hepes buffer (pH 7.5) containing60 mM KCl, 5 mM MgCl₂ as well as 1 mM dithiothreitol and 10% (v/v)glycerol. The protein was analysed by SDS-Page.

The method measures the ability of putative ligands to modulate theinteraction between the purified bacterial expressed FXR ligand bindingdomain (LBD) and a synthetic biotinylated peptide based on residues676-700 of SRC-1 (LCD2, 676-700). The sequence of the peptide used wasB-CPSSHSSLTERHKILHRLLQEGSPS-COOH where the N-terminus was biotinylated(B). The ligand binding domain (LBD) of FXR was expressed as fusionprotein with GST in BL-21 cells using the vector pDEST15. Cells werelysed by sonication, and the fusion proteins purified over glutathionesepharose (Pharmacia) according to the manufacturers instructions. Forscreening of compounds for their influence on the FXR-peptideinteraction, the Perkin Elmer LANCE technology was applied. This methodrelies on the binding dependent energy transfer from a donor to anacceptor fluorophor attached to the binding partner of interest. Forease of handling and reduction of background from compound fluorescenceLANCE technology makes use of generic fluorophore labels and timeresolved detection Assays were done in a final volume of 25 μL in a 384well plate, in a Tris-based buffer (20 mM Tris-HCl pH 7.5; 60 mM KCl, 5mM MgCl₂; 35 ng/μL BSA), containing 20-60 ng/well recombinantlyexpressed FXR-LBD fused to GST, 200-600 nM N-terminally biotinylatedpeptide, representing SRC1 aminoacids 676-700, 200 ng/wellStreptavidin-xIAPC conjugate(Prozyme) and 6-10 ng/well Eu W1024-antiGST(Perkin Elmer). DMSO content of the samples was kept at 1%. Aftergeneration of the assay mix and diluting the potentially FXR modulatingligands, the assay was equilibrated for 1 h in the dark at rt inFIA-plates black 384 well (Greiner). The LANCE signal was detected by aPerkin Elmer VICTOR2VTM Multilabel Counter. The results were visualizedby plotting the ratio between the emitted light at 665 and 615 nm. Abasal level of FXR-peptide formation is observed in the absence of addedligand. Ligands that promote the complex formation induce aconcentration-dependent increase in time-resolved fluorescent signal.Compounds which bind equally well to both monomeric FXR and to theFXR-peptide complex would be expected to give no change in signal,whereas ligands which bind preferentially to the monomeric receptorwould be expected to induce a concentration-dependent decrease in theobserved signal.

To assess the inhibitory potential of the compounds, EC₅₀-values weredetermined for example compounds as listed below in Table 1 (A=EC₅₀<25nM; B=25≦EC₅₀<100 nM; C=EC₅₀≧100 nM).

TABLE 1 Group Example # A 4, 8, 10, 13/8, 13/9, 14/1, 14/2 B 1, 2, 5, 6,8A, 13/1, 13/3, 13/4, 13/5, 13/7 C 13/2, 13/6

Mammalian One Hybrid (M1H) Assay

Determination of a ligand mediated Gal4 promoter driven transactivationto quantify ligand binding mediated activation of FXR was performed asfollows: The cDNA part encoding the FXR ligand binding domain was clonedinto vector pCMV-BD (Stratagene) as a fusion to the yeast GAL4 DNAbinding domain under the control of the CMV promoter. The amino acidboundaries of the ligand binding domain were amino acids 187-472 ofDatabase entry NM_(—)005123 (RefSeq). The plasmid pFR-Luc (Stratagene)was used as the reporter plasmid, containing a synthetic promoter withfive tandem repeats of the yeast GAL4 binding sites, driving theexpression of the Photinus pyralis (American firefly) luciferase gene asthe reporter gene. In order to improve experimental accuracy the plasmidpRL-CMV (Promega) was cotransfected. pRL-CMV contains the constitutiveCMV promoter, controlling the expression of the Renilla reniformisluciferase. All Gal4 reporter gene assays were done in HEK293 cells(obtained from DSMZ, Braunschweig, Germany) grown in MEM withL-Glutamine and Earle's BSS supplemented with 10% fetal bovine serum,0.1 mM nonessential amino acids, 1 mM sodium pyruvate, and 100 unitsPenicillin/Streptavidin per mL at 37° C. in 5% CO₂. Medium andsupplements were obtained from Invitrogen. For the assay, 5 x 10⁵ cellswere plated per well in 96 well plates in 100 μL per well MEM withoutPhenol Red and L-Glutamine and with Earle's BSS supplemented with 10%charcoal/dextran treated FBS (HyClone, South Logan, Utah), 0.1 mMnonessential amino acids, 2 mM glutamine, 1 mM sodium pyruvate, and 100units Penicillin/Streptavidin per mL, incubated at 37 ° C. in 5% CO₂.The following day the cells were >90% confluence. Medium was removed andcells were transiently transfected using 20 μL per well of aOptiMEM—polyethylene-imine-based transfection-reagent (OptiMEM,Invitrogen; Polyethyleneimine, Aldrich Cat No. 40,827-7) including thethree plasmids described above. MEM with the same composition as usedfor plating cells was added 2-4 h after addition of transfectionmixture. Then compound stocks, prediluted in MEM were added (finalvehicle concentration not exceeding 0.1%). Cells were incubated foradditional 16 h before firefly and renilla luciferase activities weremeasured sequentially in the same cell extract using aDual-Light-Luciferase-Assay system (Dyer et al., Anal. Biochem. 2000,282, 158-161). All experiments were done in triplicates.

To assess the FXR agonistic potency of the example compounds, potencyranges were determined in the M1H assay as listed below in Table 2(A=EC₅₀<25 nM; B=25≦EC₅₀<100 nM; C=EC₅₀≧100 nM).

TABLE 2 Group Example # A 13/4, 13/5, 13/6 B 2, 8, 8A, 10, 13/1, 13/3,13/7 C 1, 4, 5, 6, 13/2, 13/8, 13/9, 14/1

Aqueous Solubility Assay

The aq. solubility in PBS, pH 7.4 was determined as follows. A 10 mMcompound stock solution in DMSO was added to PBS (pH 7.4) to reach atheoretical final concentration of 200 μM. The resultingsolution/suspension was shaken at 1250 rpm for 1 h and then stored inthe dark at rt for 23 h. At this time any precipitate is separated fromthe solution by centrifugation at 3900 rpm for 30 min. The aq.solubility was determined by comparing the peak area of the principlepeak in a calibration standard (200 μM) in an organic solvent(methanol/water 60:40, v/v) with the peak area of the corresponding peakin the buffer sample. As detection method was used HPLC-UV/VIS at 230nm.

Parallel Artificial Membrane Permeation Assay (PAMPA)

For the PAMPA, 5 mM stock solutions of test items were prepared in DMSO.5 mM stock solutions of reference items were prepared in EtOH(carbamazepine, guanabenz) or in EtOH:H₂O 1:1 (v/v) (ceftriaxone),respectively. Compounds were diluted in PBS (pH 7.4) to obtain thestarting solutions containing 5% of the respective organic solvent and250 μM reference compounds or 10 μM test items, respectively. For theassay, a modified procedure of the PAMPA as described by Kansy et al.Kansy et al. (J. Med. Chem. 1998, 41, 1007) was used. The referencecompounds for low (ceftriaxone), medium (guanabenz) and high permeation(carbamazepine) were included as internal controls.

Permeation experiments were carried out in a Multiscreen 96 well tray(donor) covered by a 96-well Multiscreen Immobilon (acceptor). Thehydrophobic filter material of the Immobilon plate was pre-wetted with70% ethanol and treated with a solution of lipids (lecithin dissolved indodecane). The donor plate was filled with test compounds and referencecompounds and both plates were inserted into each other and placed ontoan orbital shaker for 15 min at 100 rpm. The transport study was startedby applying 150 μL PBS-buffer containing the test and referencecompounds to the donor plate. After 15-16 h of diffusion at rt, thecontents of the acceptor and donor plate were collected and quantifiedusing LC/MS-detection (test items) or by UV spectroscopy using aSpectramax Plus³⁸⁴ (Molecular Devices) (reference items). The absorptionmaxima for the reference items ceftriaxone, guanabenz and carbamazepinewere 240 nm, 270 nm and 286 nm, respectively. Recovery samples wereprepared as described for the permeation assay samples and wereincubated in representative vials during the permeation period under thesame conditions.

For LC/MS analysis of the test items, 100 μL incubate were removed fromacceptor and donor compartment and processed for acetonitrile (ACN)precipitation as described below. Additionally, test item samples fromthe lipid layer were extracted by flushing each well two times with 150μL EA. The solutions were collected in 1.5 mL reaction tubes and thesolvent was evaporated. The dried residues were resuspended in aPBS/DMSO/ACN mixture reflecting the composition of the acceptor anddonor samples (i.e. 100 μL buffer supplemented with 5% DMSO, 200 μLACN+ISTD). The final solvent content of each sample was 66% ACN.

Samples from donor and acceptor compartments and calibration standardswere precipitated by addition of 200 μL ACN/ISTD or 400 μL ACN/ISTD,respectively. After vigorous shaking (10 seconds) and centrifugation (5min at 4800×g, rt), the particle free supernatants were subjected toLC-MS/MS. Membrane compartments were extracted as described above. Afterreconstitution, samples were vigorously shaken (10 seconds) and spundown (5 min at 4800×g, rt). The particle free supernatants weresubjected to LC-MS/MS.

For analysis of compounds under the present invention, the HPLC systemconsisted of an Accela U-HPLC pump and an Accela auto sampler (ThermoFisher Scientific, USA). Mass spectrometry was performed on an Exactivemass spectrometer (orbitrap technology with accurate mass) equipped withan heated electrospray (H-ESI2) interface (Thermo Fisher Scientific,USA) connected to a PC running the standard software Xcalibur 2.1.

The LC was performed in the gradient mode (Table 3) using ACN/0.1%formic acid as organic phase (A) and 10 mM ammonium formate/0.1% formicacid as aq. phase (B); and the pump flow rate was set to 500 μL/min.Separation was performed on a Gemini C6-Phenyl, 3 μm, 50×2.0 mm(Phenomenex, Germany) analytical column with a pre-column (GeminiC6-Phenyl, 3 μm, 4×2.0 mm).

TABLE 3 HPLC gradients Mobile phase 0 min 0.1 min 1.2 min 2.6 min 2.7min 3.5 min A (%) 5 5 97 97 5 5 B (%) 95 95 3 3 95 95

As MS tune file a generic tune file was used for all analytes applyingthe positive or negative ion mode. As lock mass for internal masscalibration the [M+H]⁺ ion of diisooctyl phthalate(m/z 391.28429), whichis ubiquitously present in the solvent system, was used.

Analyte was acquired by scanning ±1 Thomson around the expected mass ofthe monoisotopic [M+H]⁺ or [M−H]⁻ ion. The mass resolution of theOrbitrap was set to 50,000. The accurate mass of each analyte was usedfor peak integration. Further instruments settings were as follows:HCD-Gas off, AGC high dynamic range, max. trap injection time 100 ms,sheath gas 30, aux gas 8, sweep gas 2, spray voltage 4 kV, capillarytemperature 250° C., ESI 2 heater temperature 250° C.

The objective of the present invention was to generate FXR-agonists withimproved physico-chemical properties compared to compounds claimed in WO2011/020615. This was achieved by the introduction of a polar hydroxylgroup on a 1,3-cyclobutylidene or 1,3-azetidinylidene group replacingthe former 1,2-cyclopropylidene ring.

Surprisingly, the resulting compounds maintained their activity on theFXR receptor but demonstrated improved physico-chemical properties, suchas higher aq. solubility and/or membrane permeability. A directcomparison of the corresponding compounds of the two series is given inTable 4.

TABLE 4 Aqueous PAMPA, clogD solubility Membrane permeability (ChemAxonStructure (PBS, pH 7.4) in % Flux* software)

 20 μM 13.6 5.1

192 μM 24.0 4.4

195 μM n.d.** 4.4

 72 μM 20.7 5.2

192 μM 21.1 4.5

158 μM 28.9 4.2

171 μM 46.1 3.5 *Flux (%) = (c acceptor well/sum (c donor well + cacceptor well) × 100 × 2 **n.d. = not determined

In each case either the aqueous solubility or the PAMPA membranepermeability or both are significantly improved by the introduction ofthe hydroxy-cyclobutyl or hydroxy-azetidyl moiety. As most nuclearreceptor active molecules, FXR agonists are generally very lipophilic(M. L. Crawley, Expert Opin. Ther. Patents 2010, 20, 1047). Therefore,better aqeous solubility and membrane permeability are supposed toresult in a higher oral bioavailability and in general in a bettersuitability for clinical development of those compounds as drugs (L.Huang, J. Dong, S. Karki in Evaluation of drug candidates forpreclinical development (Eds. C. Han, C. B. Davis, B. Wang), Wiley &Sons, Hoboken 2010, 187-217).

1. A compound according to the following Formula (1), an enantiomer,diastereomer, tautomer, solvate, prodrug or pharmaceutical acceptablesalt thereof

wherein R is selected from the group consisting of COOR₆, CONR₇R₈,tetrazolyl, SO₂NR₇R₈, C₁₋₆ alkyl, SO₂—C₁₋₆ alkyl and H, with R₆independently selected from the group consisting of H or C₁₋₆ alkyl, andR₇ and R₈ independently from each other selected from the groupconsisting of H, C₁₋₆ alkyl, halo-C₁₋₆ alkyl, C₁₋₆ alkylene-R₉, SO₂—C₁₋₆alkyl, wherein R₉ is selected from the group consisting of COOH, OH andSO₃H; A is selected from the group consisting of phenyl, pyridyl,pyrimidyl, pyrazolyl, indolyl, thienyl, benzothienyl, indazolyl,benzisoxazolyl, benzofuranyl, benzotriazolyl, furanyl, benzothiazolyl,thiazolyl, oxadiazolyl, each optionally substituted with one or twogroups independently selected from the group consisting of OH, O—C₁₋₆alkyl, O-halo-C₁₋₆ alkyl, C₁₋₆ alkyl, halo-C₁₋₆ alkyl, C₃₋₆ cycloalkyland halogen; Q is selected from the group consisting of phenyl, pyridyl,thiazolyl, thiophenyl, pyrimidyl, each optionally substituted with oneor two groups independently selected from the group consisting of C₁₋₆alkyl, halo-C₁₋₆ alkyl, halogen and CF₃; Y is selected from N or CH; Zis selected from

wherein X═CH, N, NO; R₁ is selected from the group consisting ofhydrogen, C₁₋₃ alkyl, C₃₋₆ cylcoalkyl, C₄₋₅ alkylcycloalkyl, whereinC₁₋₃ alkyl is optionally substituted with 1 to 3 substituentsindependently selected from halogen, hydroxy or C₁₋₆ alkoxy; R₂ and R₃are independently selected from the group consisting of hydrogen, C₁₋₃alkyl, C₁₋₃ haloalkyl, C₁₋₃ alkoxy, C₁₋₃ haloalkoxy and halogen.
 2. Thecompound according to claim 1 wherein R-A is selected from


3. The compound according to claim 1 wherein Q is


4. The compound according to claim 1 wherein Z is


5. The compound according to claim 1 selected from


6. A compound according to claim 1 for use as a medicament.
 7. Acompound according to claim 1 for use in the prophylaxis and/ortreatment of diseases mediated by FXR.
 8. The compound for use accordingto claim 7 wherein the disease is selected from chronic intrahepatic orsome forms of extrahepatic cholestatic conditions; liver fibrosis;obstructive or chronic inflammatory disorders of the liver; livercirrhosis; liver steatosis and associated syndromes, cholestatic orfibrotic effects that are associated with alcohol-induced cirrhosis orwith viral-borne forms of hepatitis; liver failure or liver ischemicafter major liver resection; chemotherapy associated steatohepatitis(CASH); acute liver failure; and/or Inflammatory Bowel Diseases.
 9. Thecompound for use according to claim 7 wherein the disease is selectedfrom lipid and lipoprotein disorders; Type Il Diabetes and clinicalcomplications of Type I and Type II Diabetes, including diabeticnephropathy, diabetic neuropathy, diabetic retinopathy and otherobserved effects of clinically manifest long term Diabetes; conditionsand diseases which result from chronic fatty and fibrotic degenerationof organs due to enforced lipid and specifically triglycerideaccumulation and subsequent activation of profibrotic pathways, such asNon-Alcoholic Fatty Liver Disease (NAFLD), or Non-AlcoholicSteatohepatitis (NASH); obesity or metabolic syndrome (combinedconditions of dyslipidemia, diabetes or abnormally high body-massindex); and/or acute myocardial infarction, acute stroke or thrombosiswhich occurs as an endpoint of chronic obstructive atherosclerosis. 10.The compound for use according to claim 7 wherein the disease isselected from non-malignant hyperproliferative disorders and malignanthyperproliferative disorders, specifically of hepatocellular carcinoma,colon adenoma and polyposis, colon adenocarcinoma, breast cancer,pancreas adenocarcinoma, Barrett's esophagus or other forms ofneoplastic diseases of the gastrointestinal tract and the liver.
 11. Useof a compound according to claim 1 for the preparation of a medicamentfor the prophylaxis and/or treatment of diseases mediated by FXR. 12.The use according to claim 11 the disease is selected from chronicintrahepatic or some forms of extrahepatic cholestatic conditions; liverfibrosis; obstructive or chronic inflammatory disorders of the liver;liver cirrhosis; liver steatosis and associated syndromes, cholestaticor fibrotic effects that are associated with alcohol-induced cirrhosisor with viral-borne forms of hepatitis; liver failure or liver ischemiaafter major liver resection; chemotherapy associated steatohepatitis(CASH); acute liver failure; and/or Inflammatory Bowel Diseases.
 13. Theuse according to claim 11 wherein the disease is selected from lipid andlipoprotein disorders; Type Il Diabetes and clinical complications ofType I and Type Il Diabetes, including diabetic nephropathy, diabeticneuropathy, diabetic retinopathy and other observed effects ofclinically manifest long term Diabetes; conditions and diseases whichresult from chronic fatty and fibrotic degeneration of organs due toenforced lipid and specifically triglyceride accumulation and subsequentactivation of profibrotic pathways; such as Non-Alcoholic Fatty LiverDisease (NAFLD) or Non-Alcoholic Steatohepatitis (NASH); obesity ormetabolic syndrome (combined conditions of dyslipidemia, diabetes orabnormally high body-mass index); and/or acute myocardial infarction,acute stroke or thrombosis which occurs as an endpoint of chronicobstructive atherosclerosis.
 14. The use according to claim 11 whereinthe disease is selected from non-malignant hyperproliferative disordersand malignant hyperproliferative disorders, specifically ofhepatocellular carcinoma, colon adenoma and polyposis, colonadenocarcinoma, breast cancer, pancreas adenocarcinoma, Barrett'sesophagus or other forms of neoplastic diseases of the gastrointestinaltract and the liver.